Fatty-acid amide hydrolase

ABSTRACT

The soporific activity of cis-9,10-octadecenoamide and other soporific fatty acid primary(amides is neutralized by hydrolysis in the presence of fatty-acid amide hydrolase (FAAH). Hydrolysis of cis-9,10-octadecenoamide by FAAH leads to the formation of oleic acid, a compound without soporific activity. FAAH has be isolated and the gene encoding FAAH has been cloned, sequenced, and used to express recombinant FAAH. Inhibitors of FAAH are disclosed to block the hydrolase activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 08/489,535, filed on Jun. 12, 1995, now abandoned, the disclosures of which is incorporated herein by reference.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under a National Institutes of Health Shared Instrumentation grant No. 1 S10 RR07273-01. The government has certain rights in the invention.

TECHNICAL

The invention relates to an enzyme which catalyzes a hydrolytic conversion between soporific fatty acid primary amides and their corresponding fatty acids and is designated a fatty-acid amide hydrolase (FAAH), to methods for enzymatically catalyzing such conversions, and to methods for inhibiting the enzymatic catalysis of such conversions. More particularly, the invention relates to FAAH protein, in either isolated or recombinant form, and to its use and inhibition.

BACKGROUND

Sleep is a natural, periodic behavioral state during which the body rests itself and its physiological powers are restored. It is characterized by a loss of reactivity to the environment. During sleep, certain physiological processes of both the body and the brain function differently than they do during alert wakefulness. Normal sleep consists of at least two quite different behavioral states: synchronized sleep, during which the electroencephalogram consists of slow waves of high amplitude, and desynchronized sleep (DS) or activated sleep characterized by rapid eye movements (REM sleep), in which the electroencephalogram pattern is characterized by waves of high frequency and low amplitude. Synchronized sleep is further characterized by slow and regular respiration, by relatively constant heart rate and blood pressure, and by a predominance of delta waves. Synchronized sleep usually consists of four stages, followed by a period of activated sleep. Each cycle lasts between 80 and 120 minutes. In contrast, desynchronized sleep is further characterized by irregular heart rate and respiration, periods of involuntary muscular jerks and movements, and a higher threshold for arousal. Periods of desynchronized sleep last from 5-20 minutes and occur at about 90 minute intervals during a normal night's sleep.

Sleep disorders include sleep deprivation and paroxysmal sleep, i.e., narcolepsy. There has been no known pharmacological method for promoting or inhibiting the initiation of sleep or for maintaining the sleeping or waking state.

Cerebrospinal fluid (liquor cerebrosinalis) is a clear, colorless fluid that circulates within the four ventricles of the brain and the subarachnoid spaces surrounding the brain and spinal cord Cerebrospinal fluid originates as an ultrafiltrate of the blood secreted by the choroid plexus in the lateral third and fourth ventricles. Cerebrospinal fluid is also sometimes called neurolymph. After passing through the four ventricles and the subarachnoid spaces, cerebrospinal fluid is largely resorbetd into the venous system via the arachnoid villi. Cerebrospinal fluid serves as a medium for the removal of catabolites, excretions, and waste materials from the tissues bathed by it. To date, no factor derived from cerebrospinal fluid has been reported to correlate with sleep deprivation. What is needed is a method for analyzing cerebrospinal fluid for identifying a biochemical factor generated by subject that correlates with sleep deprivation.

Since the seminal discovery of prostaglandins, there has been increasing recognition of the role of fatty acids and their derivatives in important physiological processes, e.g., B. Samuelsson, Les Prix Nobel1982, pp. 153-174.

Cis-9,10-Octadecenoamide has been isolated from the cerebrospinal fluid of sleep-deprived cats and has been shown to exhibit sleep-inducing properties when injected into rats. Other fatty acid primary amides in addition to cis-9,10-octadecenoamide were identified as natural constituents of the cerebrospinal fluid of cat, rat, and man, indicating that these compounds compose a distinct family of brain lipids. Together, these results teach that fatty acid primary amides represent a new class of biological signalling molecules that can be employed for inducing subjects to sleep. Preferrers fatty acid primary amides include an alkyl chain having an unsaturation and are represented by the following formula: NH₂C (O) (CH₂)_((6≧n≦11))CH═CH(CH₂)_((8≧n≦5))CH₃. Preferred soporific fatty acid primary amides have an unsaturation with a cis configuration within their alkyl chain. In addition to cis-9,10-octadecenoamide, other soporifically active fatty acid primary amides include cis-8,9-octadecenoamide, cis-11,12-octadecenoamide, and cis-13,14-docosenoamide.

Deutsch et al, Biochem. Pharmacol., 46:791 (1993) has identified an amidase activity which catalyzes both the hydrolysis and synthesis of arachidonylethanolamide (anandamide) from the membrane subcellular fractions taken from neuroblastoma, glioma cells and crude homogenates of rat brain tissues. The study detected the uptake and enzymatic breakdown of arachidonylethanolamide (anandamide) to arachidonic acid (and vice versa) from the homogenates of tissues from brain, liver, kidney and lung but not from rat heart and skeletal muscles.

The active membrane fraction which displayed this amidase activity was prepared by either homogenizing the desired cell line and subsequently subjecting the crude homogenate to density centrifugation or by taking the crude homogenates of rat brains and directly incubating them with anandamide.

The uptake and degradation of arachidonylethanolamide (anandamide) was assayed by incubation of [³H]-anandamide (NEN, NET-1073, 210 Ci/mmol) in the cell culture medium. It was found, by liquid scintillation counting of the aqueous and organic phases, that arachidonic acid and anaiidamide distributed in the organic phase. Thus, the organic extract of the cell medium was subsequently visualized using thin-layer chromatography, sprayed with a surfaces autoradiograph enhancer (EN³HANCE, Dupont) and exposed to X-ray film (Kodak X-OMAT AR) at −80° C.

The serine protease inhibitor, phenylmethylsulfonyl fluoride at 1.5 mM concentration completely inhibited the amidase activity. Other inhibitors tested had little or no effect on the activity and included aprotinin, benzamidine, leupeptin, chymostatin and pepstatin.

In a second manuscript, Deusch et. al. (J. Biol Chem., 1994, 269, 22937) reports the synthesis of several types of specific inhibitors of anandamide hydrolysis and their ability to inhibit anandamide breakdown in vitro. Four classes of compounds were synthesized and include fatty acyl ethanolamides, α-keto ethanolamides, α-keto ethyl esters and trifluoromethyl ketones. The most effective class of compounds were the trifluoromethyl ketones and α-keto esters. The least potent inhibitors were the α-keto amides and saturated analogs of anandamide.

As an example, when anandamide is incubated with neuroblastoma cells, it is rapidly hydrolyzed to arachidonate but in the presence of the inhibitor arachidonyl trifluoromethyl ketone, there is a 5 fold increase of anandamide levels. The study infers that polar carbonyls such as those found in trifluoromethyl ketones, may form stabilized hydrates that mimic the tetrahedral intermediates formed during the reaction between the nucleophilic residue and the carbonyl group of anandamide. Deutsch suggests that the nucleophilic residue may be the active site of a serine hydroxyl in the hydrolytic enzyme.

This enzyme is classified as an amidase (EC #3.5) where the enzyme acts on carbon nitrogen bonds other than peptide bonds. The amidase activity is inhibited by the serine protease inhibitor, PMSF and the action of trifluoromethyl ketone inhibitors (and others) directly affect the hydrolytic activity of the enzyme. Furthermore, Deutsch suggests that anandamide is cleaved by a mechanism that involves an active site serine hydroxyl group.

What is needed is an identification of enzymes within the brain tissue which catalyze the degradation of soporific compound found in the cerebrospinal, for mediating the soporific activity of these compounds. What is needed is an identification of inhibitors for inhibiting the activity of enzymes which degrade soporific compounds of the type found in cerebrospinal fluid.

BRIEF SUMMARY OF THE INVENTION

An enzyme is disclosed herein which degrades soporific fatty acid primary amides, and is designated fatty-acid amide hydrolase, or FAAH. FAAH is one of the enzymes which mediates the activity of fatty acid primary amides, including soporific fatty acid primary amides.

As disclosed herein, FAAH is characterized by an enzymic activity for catalyzing a conversion cis-9,10-octadecenoamide to oleic acid, among other substrates, as shown in Scheme 1 below, and therefor was originally identified as cis-9,10-octadecenoamidase. However, it is now shown that FAAH has activity to hydrolyse a variety of fatty acid primary amides, and therefore the amidase originally referred to as cis-9,10-octadecenoamidase is more appropriately reffered to as FAAH.

One aspect of the invention is directed to a purified form of FAAH. FAAH can be purified by a variety of methods, including a chromatographic methodology. Preferred chromatographic methodologies include affinity chromatography, electric chromatography, gel filtration chromatography, ion exchange chromatography, and partition chromatography. In affinity chromatography, a solid phase adsorbent contains groups that bind particular proteins because they resemble ligands for which the proteins have a natural affinity. In a preferred mode, the solid phase adsorbent contains one or more FAAH inhibitors which bind the enzyme. In antibody affinity chromatography, a solid phase immunoabsorbent having antibodies with a bind specificity with respect to FAAH are employed. In electric chromatography or electrophoresis, the FAAH is separated from other molecules according to its molecular weight or isoelectric point. In gel filtration, also known as gel permeation, molecular sieve, and exclusion chromatography, the solid phase creates a stationary phase of gel solvent and a mobile phase of excluded solvent. The FAAH is separated according to its molecular size as it partitions between the stationary and mobile phases. The gel particles are selected to have a exclusion size in excess of FAAH. In ion exchange chromatography, a solid phase ion exchanger is employed for separating the FAAH from other molecules according to its partitioning between ionic and nonionic forces. In partition chromatography, immiscible fluids having a stationary and mobile phases are employed for separating the FAAH according to its partitioning between the two immiscible phases. Preferred chromatographic methodologies include DEAE chromatography, affinity chromatography on a solid phase having attached Hg groups derivatized with an inhibitor of FAAH such as a trifluoroketone.

In a preferred mode, a crude source of FAAH is purified in four steps. In the first step, a crude source of FAAH is purified by exchange chromatography using a DEAE chromatography column to form a first elution product. In the second step, the elution product from the first step is further purified by partitioning by with affinity chromatography to form a second elution product. In the third step, elution product from the second step is further purified by partitioning with Heparin affinity chromatography to form a third elution product. In the fourth step, the elution product from the third step is further purified by partitioning with an stationary phase derivatized with a trifluoroketone inhibitor of FAAH. The eluant from the fourth step form the purified form of FAAH. FAAH can be isolated from any of a variety of mammalian species, including rat, mouse or human, as described herein.

Fatty-acid amid hydrolase (FAAH) is characterized by inclusion of an amino acid sequence selected from a group consisting of:

a.) GGSSGGEGALIGSGGSPLGLGTDIGGSIRFP (SEQ ID NO 5), b.) SPGGSSGGEGALIGS (SEQ ID NO 6), c.) ALIGSGGSPLGLGTD (SEQ ID NO 7), d.) GLGTDIGGSIRFPSA (SEQ ID NO 8), e.) RFPSAFCGICGLKPT (SEQ ID NO 9), f.) GLKPTGNRLSKSGLK (SEQ ID NO 10), g.) KSGLKGCVYGQTAVQ (SEQ ID NO 11), h.) QTAVQLSLGPMARDV (SEQ ID NO 12), i.) MARDVESLALCLKAL (SEQ ID NO 13), j.) CLKALLCEHLFTLDP (SEQ ID NO 14), k.) FTLDPTVPPFPFREE (SEQ ID NO 15), l.) PFREEVYRSSRPLRV (SEQ ID NO 16), m.) RPLRVGYYETDNYTM (SEQ ID NO 17), n.) DNYTMPSPAMRRALI (SEQ ID NO 18), o.) RRALIETKQRLEAAG (SEQ ID NO 19), p.) LEAAGHTLIPFLPNN (SEQ ID NO 20), q.) FLPNNIPYALEVLSA (SEQ ID NO 21), r.) EVLSAGGLFSDGGRS (SEQ ID NO 22), s.) DGGRSFLQNFKGDFV (SEQ ID NO 23), t.) KGDFVDPCLGDLILI (SEQ ID NO 24), u.) DLILILRLPSWFKRL (SEQ ID NO 25), v.) WFKRLLSLLLKPLFP (SEQ ID NO 26), w.) KPLFPRLAAFLNSMR (SEQ ID NO 27), x.) LNSMRPRSAEKLWKL (SEQ ID NO 28), y.) KLWKLQHEIEMYRQS (SEQ ID NO 29), z.) MYRQSVIAQWKAMNL (SEQ ID NO 30), aa.) KAMNLDVLLTPMLGP (SEQ ID NO 31), and ab.) PMLGPALDLNTPGR (SEQ ID NO 32).

Another aspect of the invention is directed to a method for catalyzing the hydrolysis of a fatty acid primary amide. In this hydrolysis method, the fatty acid primary amide is combined or contacted with a catalytic amount of purified form of FAAH. In a preferred mode, the fatty acid primary amide is of a type which includes an alkyl chain having an unsaturation or more particularly is represented by the following formula:

NH₂C(O)(CH₂)_((6≧n≦11))CH═CH(CH₂)_((8≧n≦5))CH₃,

More particularly, the unsaturation of the alkyl chain may have a cis configuration or may be identically cis-9,10-octadecenoamide, cis-8,9-octadecenoamide, cis-11,12-octadecenoamide, or cis-13,14-docosenoamide.

Another aspect of the invention is directed to a method for inhibiting an enzymatically catalyzed hydrolysis of fatty acid primary amides, such as cis-9,10-octadecenoamide, by FAAH. In this method, FAAH is combined or contacted with an inhibitor of FAAH. Preferred inhibitors include phenylmethylsulfonyl fluoride, HgCl₂, and a trifluoroketone having the following structure:

Another aspect of the invention is directed to a method for ascertaining the inhibitory activity of a candidate inhibitor of FAAH. Thus, FAAH is admixed with a candidate FAAH inhibitor to assess inhibitory capacity in a screening method.

In a preferred method for determining inhibitory activity of a candidate FAAH inhibitor, the contemplated method comprises five steps. In the first step, a mixture “A” is formed by combining FAAH and cis-9, 10-octadecenoamide substrate under reaction conditions. In the second step, a mixture “B” is formed by combining the mixture “A” with the candidate inhibitor. In the third step, the conversion of cis-9,10-octadecenoamide substrate to a hydrolysis product within mixture “A” is quantified. In the fourth step, the conversion of cis-9,10-octadecenoamide substrate to hydrolysis product within mixture “B” is quantified. In the fifth step, the inhibitory activity of the candidate inhibitor is ascertained by comparing the quantifications of steps three and four.

Another aspect of the invention is directed to a trifluoroketone inhibitor of FAAH represented by following structure:

Another aspect of the invention is directed to one or more nucleotide sequences the encode part or all of FAAH. The complete nucleotide sequence that encodes human, mouse or rat FAAH are shown in SEQ ID Nos. 42, 39 or 35, respectively.

The partial nucleotide sequence of rat FAAH is represented as follows:

CCAGGAGGTTCCTCAGGGGGTGAGGGGGCTC (SEQ ID NO 54) TCATTGGATCTGGAGGTTCCCCTCTGGGTTT AGGCACTGACATTGGCGGCAGCATCCGGTTC CCTTCTGCCTTCTGCGGCATCTGTGGCCTCA AGCCTACTGGCAACCGCCTCAGCAAGAGTGG CCTGAAGGGCTGTGTCTATGGACAGACGGCA GTGCAGCTTTCTCTTGGCCCCATGGCCCGGG ATGTGGAGAGCCTGGCGCTATGCCTGAAAGC TCTACTGTGTGAGCACTTGTTCACCTTGGAC CCTACCGTGCCTCCCTTTCCCTTCAGAGAGG AGGTCTATAGAAGTTCTAGACCCCTGCGTGT GGGGTACTATGAGACTGACAACTATACCATG CCCAGCCCAGCTATGAGGAGGGCTCTGATAG AGACCAAGCAGAGACTTGAGGCTGCTGGCCA CACGCTGATTCCCTTCTTACCCAACAACATA CCCTACGCCCTGGAGGTCCTGTCTGCGGGCG GCCTGTTCAGTGACGGTGGCCGCAGTTTTCT CCAAAACTTCAAAGGTGACTTTGTGGATCCC TGCTTGGGAGACCTGATCTTAATTCTGAGGC TGCCCAGCTGGTTTAAAAGACTGCTGAGCCT CCTGCTGAAGCCTCTGTTTCCTCGGCTGGCA GCCTTTCTCAACAGTATGCGTCCTCGGTCAG CTGAAAAGCTGTGGAAACTGCAGCATGAGAT TGAGATGTATCGCCAGTCTGTGATTGCCCAG TGGAAAGCGATGAACTTGGATGTGCTGCTGA CCCCNATGYTNGGNCCNGCNYTNGAYYTNAA YACNCCNGGNMGN

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structures of natural agent, cis-9,10-octadecenoamide (1), related analogs (2-6). Compound 6 is the preferred structure for naturally occurring C₂₂ fatty acid amide.

FIG. 2 illustrates the determined partial amino acid sequence of the rat FAAH as described in Section B.4.

FIG. 3 illustrates a partial purification strategy involving isolation of a plasma membrane protein fraction from rat liver using 1) a sucrose gradient of the liver membrane followed by 2) a 100 mM sodium carbonate wash and 3) solubilization in trion-based buffer. The isolated liver plasma membrane is then purified by four consecutive chromatographic steps: 1) Ion exchane DEAE column, 2) Mercury inhibition column, 3) detergent exchange Heparin column followed by 4) an affinity column with a trifluoroketone inhibitor. The purified protein was determined to have a 20-30 fold enrichment of amidase activity from crude membrane protein fraction by visual comparison of the purified protein band intensity with the crude protein fraction. Estimated purified yield is 10-15% from crude liver plasma membrane protein.

FIG. 4 illustrates the affinity column purification strategy (step 4, FIG. 3) using a trifluoroketone inhibitor which is linked to a disulfide-derivatized solid support (pyridyl disulfide beads).

FIG. 5 illustrates the synthetic protocol for the synthesis of the trifluoroketone inhibitor and subsequent attachment of the inhibitor to the disulfide-derivatized solid support using pyridyl disulfide beads.

FIG. 6 represents an autoradiogram of a thin layer chromatography plate (SiO₂, 55% ethyl acetate/hexanes) illustrating the FAAH activity of a rat brain membrane fraction with respect to the hydrolysis of radio-labelled cis-9,10-octadecenoamide to oleic acid and its inhibition by phenylmethyl sulfonyl fluoride (PMSF). Lane number, content: lane 1, Cis-9,10-octadecenoamide standard; lane 2, Cis-9,10-octadecenoamide with rat brain soluble fraction; lane 3, Cis-9,10-octadecenoamide with rat brain membrane fraction; lane 4, Cis-9,10-octadecenoamide with rat brain membrane fraction +1 mM phenylmethylsulfonyl fluoride (PMSF); lane 5, Cis-9,10-octadecenoamide with rat brain membrane fraction +5 mM EDTA; lane 6, Cis-9,10-octadecenoamide with rat pancreatic microsomes; lane 7, Cis-9,10-octadecenoamide with proteinase K (200 mg); lane 8, oleic acid standard.

FIG. 7 represents an autoradiogram of a thin layer chromatography plate (SiO₂, 55% ethyl acetate/hexanes) illustrating the FAAH activity of a rat brain membrane fraction with respect to the hydrolysis of radio-labelled cis-9,10-octadecenoamide to oleic acid and its inhibition by mercuric chloride (HgCl₂). The optimal concentrations required for inhibition of amide hydrolysis activity lies between 50 mM and 5 mM HgCl₂. The various lanes of the TLC plate are identified as follows: lane 1, Cis-9,10-octadecenoamide standard; lane 2, Cis-9,10-octadecenoamide with rat brain soluble fraction; lane 3, Cis-9,10-octadecenoamide with rat brain membrane fraction and 500 mM HgCl₂; lane 4, Cis-9,10-octadecenoamide with rat brain membrane fraction and 50 mM HgCl₂; lane 5, Cis-9,10-octadecenoamide with rat brain membrane fraction and 5 mM HgCl₂; lane 6, oleic acid standard. A typical HgCl₂ inhibition study uses a 100 mM HgCl₂ stock (27 mg in lmL Tris buffer (50 mM), pH 7.5) of HgCl₂.

FIG. 8 represents a northern blot of mRNA obtained from cloning procedures. Ribosomal markers are shown by the arrows, next to lane 1, and indicate 5 kb and 2 kb bands. The arrow next to lane 6 points to a 3 kb band which is representative of the oleic amidase enzyme. Lane 1 is total RNA from rat brain; lane 2 is total RNA from rat lung; lane 3 is total RNA from rat kidney; lane 4 is total RNA from rat heart; lane 5 is total RNA from rat liver; lane 6 is mRNA from rat liver (mRNA loaded in lane 6 is approximately 500 ng); total respective RNA loaded in lanes 1-5 was approximately 15 μg.

FIG. 9 illustrates the deduced encoded amino acid residue sequence of rat oleamide hydrolase also referred to as a fatty acid amide hydrolase or FAAH (SEQ ID NO: 36). The encoded rat FAAH is appropriately abbreviated rFAAH. Bold type indicates the putative transmembrane spanning domain as predicted by PSORT. The seven discontinuous underlined regions indicate the seven separate peptides, the designation of which is consecutive, obtained by HPLC purification of a trypsin digest of the enzyme. The double-underlined segment is the putative SH3-domain-binding sequence.

FIGS. 10-1 through 10-5 show the continuous double-stranded cDNA sequence for rat FAAH as described in Section D. The encoded amino acid sequence is also indicated beginning with the ATG start site encoding methionine (M). The stop codon is also shown as boxed. The top and bottom strands of the cDNA sequence are respectively listed in SEQ ID NOs 35 and 37 with the amino acid sequence listed with the nucleotide sequence in SEQ ID NO 35 and by itself in SEQ ID NO 36.

FIG. 11 illustrates the alignment of the amidase signature sequence region of the rat FAAH (SEQ ID NO 36 from amino acid residue 215 to and including 246) with several other representative amidases as further described in Section D1. Residues of the signature sequence that are completely conserved among the family members are shown in bold type and the relative amino acid position of the signature sequence of each member is given by the numbers just preceding and following the sequence information. From top to bottom, the sequences have the following respective SEQ ID NOs: 36 (from residue 215 to 246) ; 47, 48, 49, 50, 51, 52 and 53.

FIG. 12A and 12B show the respective results of Southern and Northern blots as probed with an internal 800 bp fragment of rat FAAH cDNA as further described in Section D.

FIGS. 13-1 through 13-4 show the continuous double-stranded cDNA sequence for mouse FAAH as described in Section D2. The encoded amino acid sequence is also indicated beginning with the ATG start site encoding methionine (M). The stop codon is also shown as boxed. The top and bottom strands of the cDNA sequence are respectively listed in SEQ ID NOs 39 and 41 with the amino acid sequence listed with the nucleotide sequence in SEQ ID NO 39 and by itself in SEQ ID NO 40.

FIGS. 14-1 through 14-5 show the continuous double-stranded cDNA sequence for human FAAH as described in Section D3. The encoded amino acid sequence is also indicated beginning with the ATG start site encoding methionine (M). The stop codon is also shown as boxed. The top and bottom strands of the cDNA sequence are respectively listed in SEQ ID NOs 42 and 44 with the amino acid sequence listed with the nucleotide sequence in SEQ ID NO 42 and by itself in SEQ ID NO 43.

FIG. 15A shows the expression of recombinant rat FAAH in COS-7 cells produced as described in Section E as performed by thin layer chromatography demonstrating the conversion of labeled oleamide to oleic acid as further described in Section F.

FIG. 15B shows the inhibition of recombinant rat FAAH by trifluoromethyl ketone also performed as described in FIG. 15A as further described in Section F.

FIG. 15C shows the results of Western blotting of recombinant rat FAAH with antibodies generated against peptide 2 as shown in FIG. 9 as shown in the four left lanes (1-4) and as competed with peptide 2 as shown in the four right lanes (5-8). Samples of untransfected COS-7 cell extract are shown in lanes 4 and 8, FAAH-transfected COS-7 cell extracts are shown in lanes 3 and 7, affinity-purified rat FAAH is shown in lanes 2 and 6 and a mixture of FAAH-transfected COS-7 cell extracts and affinity-purified FAAH is run in lanes 1 and 5. The proteins were probed with antibodies in the absence (lanes 1-4) or presence (lanes 5-8) of competing peptide antigen. The FAAH-transfected COS-7 cell extract but not the control contained an immunoreactive 60K-65K protein that was effectively competed away by preincubation of the antibodies with excess peptide antigen while the trace quantities of cross reactive protein observed in both transfected and untransfected COS-7 cell extracts were not competed by the peptide.

FIG. 16 shows the ability of human recombinant expressed FAAH to hydrolyze oleamide to oleic acid, as further described in FIG. 15A with thin layer chromatography and in Section F.

FIG. 17 shows the results of thin layer chromatography demonstrating the conversion of labeled anandamide to arachidonic acid in rat FAAH-transfected COS-7 cells as shown in lane 3 but not in control untransfected cells (lane 2). TLC standards of anandamide and arachidonic acid are shown in lanes 1 and 4, respectively.

DETAILED DESCRIPTION OF THE INVENTION

A. Protocols for the Induction of Sleep

Synthetic cis-9,10-octadecenoamide was injected (ip) into rats in order to test its effect on spontaneous behavior at different doses: 1 (n=2), 2 (n=2), 5 (n=7), 10 (n=10), 20 (n=2), and 50 (n=2) mg, where n =number of rats tested. Rats were injected during a reversed dark period (12:12) two hours after the lights cycled off and were observed in their home cages. With the lower doses (1 and 2 mg), no overt effect on spontaneous behavior was witnessed. However, at a threshold of 5 mg and above there was a marked effect consisting of an induction of long-lasting motor quiescence associated with eyes closed, sedated behavior characteristic of normal sleep. Also as with normal sleep, the rats still responded to auditory stimuli with orienting reflex and sustained attention toward the source of stimulation. In addition, motor behavior was impaired. The latency to behavioral sedation following administration was about 4 minutes and subjects were normally active again after 1 hour (5 mg), 2 hour (10 mg), or 2.5 hour (20 mg and 50 mg).

We have compared cis-9,10-octadecenoamide to vehicle and the synthetic analogs listed in FIG. 1 to estimate the structural specificity of its sleep-inducing potential. Neither vehicle (5% ethanol in saline solution) nor oleic acid (5) showed any overt behavioral effect. Trans-9,10-octadecenoamide demonstrated similar pharmacological effects to its cis counterpart, but was much less potent as measured by the comparatively shorter duration time for its sleep-inducing properties (at 10 mg per rat, the biological effect lasted one hour for the trans isomer and two hours for the cis isomer). When the olefin was moved either direction along the alkyl chain (to the 8,9 (3) or 11,12 (4) positions) or the alkyl chain length was extended to 22 carbons (6), a substantial reduction in both the degree and duration of the pharmacological effects was observed, and while the mobility of the rats still decreased, their eyes remained open and their alertness appeared only slightly affected. Finally, polysomnographic studies on rats injected with cis-9,10-octadecenoamide show an increase in the total time of slow wave sleep (SWS) as well as in the mean duration of the SWS individual periods when compared to vehicle controls. More particularly, male Sprague-Dawley rats (300 g at the time of surgery) were implanted under halothane anesthesia (2-3%) with a standard set of electrodes for sleep recordings. This included two screw electrodes placed in the parietal bone over the hippocampus to record the subjects electroencephalogram (EEG) and two wire electrodes inserted in the neck musculature to record postural tone through electromyographic activity (EMG). Rats were housed individually with at libitum access to food and water. The dark-light cycle was controlled (12:12, lights on a 10:00 p.m.). One week after the surgery, rats were habituated to the recording conditions for at least three days. Upon the completion of the habituation period, rats received 2 milliliter (ip) of either: vehicle (5% ethanol/saline solution), cis-9,10-octadecenoamide (10 mg), or oleic acid (10 mg). Rats were continuously recorded for four hours after the ip injection (12:00 p.m.-4:00 p.m.) Rats were observed for spontaneous changes in behavior through a one-way window. Sleep recordings were visually scored and four stages were determined: wakefulness, slow-wave-sleep 1 (SWS1), slow-wave-sleep 2 (SWS2), and rapid eye movement (REM) sleep.

These increases with respect to slow wave sleep (SWS) were at the expense of waking. Distribution of REM sleep does not seem to be altered. Together, these data suggest that cis-9,10-octadecenoamide could play an important role in slow-wave sleep modulation.

The traditional view of lipid molecules as passive structural elements of cellular architecture is rapidly giving way to an ever increasing awareness of the active roles these agents play in transducing cell signals and modifying cell behavior, e.g., Liscovitch et al, Cell, 77:329 (1994). An intriguing feature of the fatty acid amides studied here is that they belong to a family of simple molecules in which a great deal of diversity may be generated by simply varying the length of the alkane chain and the position, stereochemistry, and number of its olefin(s). Interestingly, other neuroactive signalling molecules with amide modifications at their carboxy termini have been reported, from carboxamide terminal peptides to arachidonylethanolamide. Neuroactive signalling molecules employing carboxamide terminal peptides are disclosed by Eipper et al, Annu. Rev. Neurosci., 15:57 (1992). Neuroactive signalling molecules employing arachidonylethanolamide is disclosed by Devane et al, Science, 258:1946 (1992). It is disclosed herein that cis-9,10-octadecenoamide is a member of a new class of biological effectors in which simple variations of a core chemical structure have unique physiological consequences.

B. Isolation and Assay of Integral Membrane Protein Fraction with FAAH Activity

1. Observations on Lipid Amidase Activity

Lipid amidase activity has been observed in brain, liver, lung, kidney and spleen tissues, but not in heart tissue. The activity is inhibited by 1 mM PMSF (phenylmethylsulfonyl fluoride) and 50 mM HgCl, which is a test for sulfhydryl group dependency of the reaction. Since the fractions are not solubilized by 100 mM sodium carbonate (pH 11.5), the sample is apparently a membrane protein, which has been identified in nuclear, microsomal, and plasma membrane subcellular fractions, but not in the cytosol.

The enzyme catalyzed hydrolysis of cis-9,10-octadecenoamide to oleic acid by purified cis-9,10-octadecenoamide and inhibition of this enzyme by PMSF is disclosed on an autoradiogram of a thin layer chromatographic plate (SiO2, 55% ethyl acetate/hexanes), illustrated in FIG. 6. In each case the enzymic reaction is performed is a separate reaction vessel and the product is spotted onto a TLC plate. The various reaction conditions for the reaction vessel corresponding to each lane are identified as follows:

lane 1: Cis-9,10-octadecenoamide standard;

lane 2: Cis-9,10-octadecenoamide with rat brain soluble fraction;

lane 3: Cis-9,10-octadecenoamide with rat brain membrane fraction;

lane 4: Cis-9,10-octadecenoamide with rat brain membrane fraction +1 mM PMSF;

lane 5: Cis-9,10-octadecenoamide with rat brain membrane fraction +5 mM EDTA;

lane 6: Cis-9,10-octadecenoamide with rat pancreatic microsomes;

lane 7: Cis-9,10-octadecenoamide with proteinase K (200 mg); and

lane 8: oleic acid standard.

Inhibition studies of Cis-9,10-octadecenoamide hydrolysis to oleic acid with HgCl₂ are illustrated in FIG. 7. Between 50 mM and 5 mM HgCl₂ lies the optimal concentrations required for inhibition of amide hydrolysis activity. The enzyme catalyzed hydrolysis of cis-9,10-octadecenoamide to oleic acid by purified cis-9,10-octadecenoamide and inhibition of this enzyme by HgCl₂ is performed in a series of reaction vessels and spotted onto a thin layer chromatographic plate (SiO2, 55% ethyl acetate/hexanes). A typical HgCl₂ inhibition study uses a 100 mM HgCl₂ stock (27 mg in lmL Tris buffer (50 mM), pH 7.5) of HgCl₂. The various reaction conditions for the reaction vessels corresponding to each lane are identified as follows:

lane 1: Cis-9,10-octadecenoamide standard;

lane 2: Cis-9,10-octadecenoamide with rat brain soluble fraction;

lane 3: Cis-9,10-octadecenoamide with rat brain membrane fraction and 500 mM HgCl₂;

lane 4: Cis-9,10-octadecenoamide with rat brain membrane fraction and 50 mM HgCl₂;

lane 5: Cis-9,10-octadecenoamide with rat brain membrane fraction and 5 mM HgCl₂;

A unique enzymatic activity capable of degrading the putative effector molecule, cis-9,10-octadecenoamide has been identified and is disclosed herein. Rapid conversion of ¹⁴C-cis-9,10-octadecenoamide to oleic acid by rat brain membrane fractions was observed by TLC. The enzymatic activity was unaffected by 5 mM EDTA, but was completely inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF). Only trace amide hydrolysis activity was observed with rat brain soluble fractions, while rat pancreatic microsomes and proteinase K showed no significant capacity to hydrolyze cis-9,10-octadecenoamide to oleic acid.

2. Synthesis of Fatty Acid Primary Amides

Preferred protocols for synthesizing exemplary fatty acid primary amides are provided. The synthetic protocols differ only with respect to the chain length of the starting materials, the product yields, and the separation of the various cis and trans products. Accordingly, exemplary descriptions of synthetic protocols for the synthesis of cis-9,10-octadecenoamide and several other fatty acid primary amides are provided and serve to illustrate the synthetic protocol for the entire class of fatty acid primary amides.

3. Isolation of Rat Integral Membrane Protein Fraction with FAAH Activity

The protocol described herein is for about 5-10 g of tissue. The rat liver(s) are collected, weighed and then placed in 1 mM NaHCO₃ on ice. Next, the liver is cut up, rinsed (2×) with 1 mM NaHCO₃ and minced with a razor blade on a sheet of wax. It is then placed into 25 ml of 1 mM sodium bicarbonate and homogenized in a tissuemizer for 2 minutes at setting 6. A dilution to 100 ml with 1 mM sodium bicarbonate is subsequently performed, which is followed by a filtration through 4 layers of cheesecloth and then through 8 layers. The filtrate is then brought up to 100 ml and split into four JA-20 tubes and topped off with 1 mM sodium bicarbonate. The tubes are spun at 6,000 rpm (4500×g) for 12 minutes at 4° C. in the JA-20 rotor. Using a Pasteur pipette, the fat layer is sucked off and the supernatant layer is decanted and saved.

Next, the pellet is resuspended in the remaining supernatant layer with a syringe and needle. 20 mL fractions of the resuspension are then dounced 16 times with a 15 ml dounce homogenizer. The fractions are then combined into a single JA-20 tube and brought up to volume with 1 mM NaHCO₃. The tubes are next spun again at 6,000 rpm (4500×g) for 15 minutes at 4° C. in a JA-20 rotor and the supernatant is subsequently poured off and saved. The pellet is resuspended and dounced as before and then brought up to 10 ml volume with lmM sodium bicarbonate. Next, 20 mL of 67% sucrose solution is added to a final volume of 30 ml and the mixture is split into 2 tubes. An additional 25 mL of 30% sucrose is added to the top of each tube and spun at 27 K rpm for 1 hour 45 minutes at 4° C. in an ultracentrifuge. The fractions are collected from the sucrose gradient and the middle band from the sucrose gradient (plasma membrane band) is placed in a capped plastic tube and filled with 1 mM sodium bicarbonate. The tube is subsequently spun at 17,000 rpm for 35 minutes at 4° C.

The supernatant is discarded and the pellets are resuspended (with Douncing) in 100 mM of sodium carbonate. This solution is subsequently kept on ice for 1 hour and then spun at 100,000 g for 1 hour. The supernatant (solubilized peripheral membrane proteins) is discarded since no lipid amidase activity is present in this fraction and the pellet is resuspended (with Douncing) in 10% glycerol, 1% Triton, 0.1% phosphatidyl choline, 20 mM Hepes buffer and then stirred for two hours at 4° C. Finally the solution is spun at 100,000 g for 1 hour and the supernatant thus obtained is further purified as follows. 4. Purification Via 4 Step Column Chromatography Process

Step 1 DEAE column/ ion exchange (FIG. 3). The above solubilized supernatant batch is further purified. The supernatant batch is mixed with DEAE-Sephadex (Diethylaminoethyl-Sephadex, commercially available from Sigma chemical company) ion exchange resin for 1 hour at 40° C. The fraction which is bound to the DEAE resin, displays the lipid amidase activity (none in flow through). Solubilized rat liver plasma membrane (in BI: 10% glycerol, 1% Triton X-100, 1 mM EDTA, 20 mM Hepes, pH 7.2) is passed over DEAE Fast Flow column (Pharmacia) and washed with 5 column volumes of BI, 0.2% Triton. Then the amidase activity is eluted with 1 column volume each of 50 mM, 100 mM, and 200 mM NaCl in BI with 0.2% Triton.

Step 2 Hg Column (FIG. 3). The above eluent from the DEAE exchange, is mixed with p-chloromercuric benzoic acid resin (Commercially available from BioRad chemical company) for 1 hour at 4° C. The fraction which is bound to the above mercury resin, displays the lipid amidase activity (none in flow through), is washed with 5 column volumes of BI with 0.2% Triton, 5 column volumes of BI with 0.2% Triton and 150 mM NaCl, and eluted with 1.5 column volumes BI with 0.2% Triton, 150 mM NaCl, and 25 mM b-mercaptoethanol.

Step 3 Heparin column (FIG. 3). Hg-eluted amidase activity was passed over Heparin column (BioRad) and washed with 10 column volumes of BI with 0.7% CHAPS and 150 mM NaCl (detergent exchange). Elution was conducted with 1 column volume each of BI with 0.7% CHAPS and 300 mM, 400 mM, 500 mM, 650 mM, and 750 mM NaCl,respectively, with amidase activity eluting in the final two fractions.

Step 4 Affinity column (FIGS. 3 and 4). Heparin-eluted amidase activity was mixed with Triton X-100 for a final concentration of 0.2%, and then passed over CF₃-inhibitor linked to activated pyridyl disulphide beads (103: attachment of inhibitor to beads is described infra) and washed with 20 column volumes of BI with 0.2% Triton X-100. Elution was conducted by passing 3 column volumes of BI with 0.2% Triton and 20 mM DTT, and letting column stand at 4o C for 30 h. Then, washing column with 1.5 column volumes of BI with 0.2% Triton and 20 mM DTT eluted single protein of 60 kD in size.

Eluted 60 kd protein was digested with trypsin and peptides were sequenced as described infra.

The purity of the activity is then assessed after this procedure according to an assay protocol.

5. Assay for Fatty-Acid Amide Hydrolase Activity:

The following thin layer chromatography (TLC) protocol is used for assaying cis-9,10 octadecenoamide hydrolysis activity, also referred to as fatty-acid amide hydrolase activity. Oleamide is first labeled with ¹⁴C. To accomplish this, ¹⁴C-Oleic acid (1-10 μM, Moravek Biochemicals, 5-50 μCi/μM) in CH₂CL₂ (200 μL, 0.005-0.05 M) at 0° C. was treated with excess oxalyl chloride and the reaction mixture was warmed to 25° C. for 6 hours. The reaction mixture was then concentrated under a constant stream of gaseous nitrogen and the remaining residue was cooled to 0° C. and treated with excess saturated aqueous ammonium hydroxide. After 5 minutes, the reaction mixture was partitioned between Et)Ac (1.5 mL) and 10% HCl (1.0 mL). The organic layer was then washed with water (1.0 mL) and concentrated under a constant stream of gaseous nitrogen to provide ¹⁴C-oleamide in quantitative yield as judged by TLC (60% EtOAc in hexanes; oleamide R_(f)-0.2; oleic acid R_(f)-0.8).

Approximately 1 μCi of ¹⁴C-oleamide (specific activity 5-50 μCi/M) in ethanol was incubated at 37° C. for 1-2 hours with 70 μL of 126 mM Tris-HCl, pH 9.0 (final concentration of ethanol was 2.0%). The reaction mixture was then partitioned between ethyl acetate (1.0 mL) and 0.07 M Hcl (0.6 mL). The ethyl acetate layer was concentrated under a constant stream of gaseous nitrogen and the remaining residue was resuspended in 15 μL of ethanol. Approximately 3 ML of this ethanol stock was then used for TLC analysis (60% EtOAc in hexanes: oleamide R_(f)-0.2; oleic acid R_(f)-0.8). Following exposure to solvent, TLC plates were air-dired, treated with EN³HANCE spray (Dupont NEN) according to manufacturer's guidelines and exposed to film at −78° C. for 1-2 hours.

The purified protein was determined to have a 20-30 fold enrichment of amidase activity from crude membrane protein fraction by visual comparison of the purified protein band intensity with the crude protein fraction. Estimated purified yield is 10-15% (FIG. 3)

C. Synthetic Protocols

1. Cis-9,10-octadecenoamide (1: FIG. 1):

A solution of oleic acid (1.0 g, 3.55 mmol, 1.0 equiv.) in CH₂Cl₂ (8.9 mL, 0.4 M) at 0° C. was treated dropwise with oxalyl chloride (5.32 mL, 2.0 M solution in CH₂Cl₂, 10.64 mmol, 3.0 equiv.). The reaction mixture was stirred at 25° C. for 4 h, concentrated under reduced pressure, cooled to 0° C., and treated with saturated aqueous NH₄OH (2.0 mL). The reaction mixture was then partitioned between ethyl acetate (EtOAc) (100 mL) and H₂O (100 mL), and the organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Chromatography (SiO₂, 5 cm×15 cm, 40-100% EtOAc-hexanes gradient elution) afforded 1 as a white solid (0.810 g, 0.996 g theoretical, 81.3%): ¹H NMR (CDCl₃, 250 MHz) δ 6.06 (bs, 1H, NH₂C(O)), 5.58 (bs, 1H, NH₂C(O)), 5.32 (m, 2H, CH═CH), 2.16 (t, 2H, J=7.5 Hz, CH₂C(O)NH₂), 2.02 (m, 4H, CH₂CH═CHCH₂), 1.61 (m, 2H, CH₂CH₂C(O)NH₂), 1.29 (b s, 14H, alkyl protons), 0.87 (t, 3H, CH₃); FABHRMS (NBA/NaI m/e 282.2804 (C₁₈H₃₅NO+H⁺ requires 282.2797). The regions of the spectra that distinguish between the cis and trans isomers are the olefinic protons from δ 5.3 to 5.2 and allylic protons from δ 2.0 to 1.8. These regions identify the natural compound as cis-9,10-octadecenoamide.

2. Trans-9,10-octadecenlamide (2: FIG. 1)

A solution of elaidic acid (1.0 g, 3.55 mmol, 1.0 equiv.) in CH₂Cl₂ (8.9 mL, 0.4 M) at 0° C. was treated dropwise with oxalyl chloride (5.32 mL, 2.0 M solution in CH₂Cl₂,10.64 mmol, 3.0 equiv.). The reaction mixture was stirred at 25° C. for 4 h, concentrated under reduced pressure, cooled to 0° C., and treated with saturated aqueous NH₄OH (2.0 mL). The reaction mixture was then partitioned between ethyl acetate (EtOAc) (100 mL) and H₂O (100 mL), and the organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, 40-100% EtOAc-hexanes gradient elution) afforded 2 as a white solid. The regions of the spectra that distinguish between the cis and trans isomers are the olefinic protons from δ 5.3 to 5.2 and allylic protons from δ 2.0 to 1.8. These regions identify the compound as trans-9,10-octadecenoamide.

3. Cis-8,9-octadecenoamide (3: FIG. 1):

A solution of 11, synthesized infra, (0.130 g, 0.461 mmol, 1.0 equiv.) in CH₂Cl₂ (1.5 mL, 0.31 M) at 0° C. was treated dropwise with oxalyl chloride (0.69 mL, 2.0 M solution in CH₂Cl₂,1.38 mmol, 3.0 equiv.). The reaction mixture was stirred at 25° C. for 4 h, concentrated under reduced pressure, cooled to 0° C., and treated with saturated aqueous NH₄OH (2.0 mL). The reaction mixture was then partitioned between ethyl acetate (EtOAc) (100 mL) and H₂O (100 mL), and the organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, 40-100% EtOAc-hexanes gradient elution) afforded 3 as a white solid. (0.105 g, 0.130 theoretical, 80.8%): ¹H NMR (CDCl₃,250 MHz) δ 5.70-5.34 (m, 4H, H₂NC(O) and CH═CH), 2.21 (t, 2H, J=7.5 Hz, CH₂C(O)NH₂), 2.00 (m, 4H, CH₂CH═CHCH₂), 1.63 (m, 2H, CH₂CH₂C(O)NH₂), 1.47-1.23 (m, 20H, alkyl protons), 0.87 (t, 3H, RCH₃); FABHRMS (NBA/CSI m/e 414.1762 (C₁₈H₃₅NO+Cs⁺ requires 414.1773).

4. Cis-11,12-octadecenoamide (4: FIG. 1):

A solution of &11,12 octadecenoic acid (1.0 g, 3.55 mmol, 1.0 equiv.) in CH₂Cl₂ (8.9 mL, 0.4 M) at 0° C. was treated dropwise with oxalyl chloride (5.32 mL, 2.0 M solution in CH₂Cl₂,10.64 mmol, 3.0 equiv.). The reaction mixture was stirred at 25° C. for 4 h, concentrated under reduced pressure, cooled to 0° C., and treated with saturated aqueous NH₄0H (2.0 mL). The reaction mixture was then partitioned between ethyl acetate (EtOAc) (100 mL) and H₂O (100 mL), and the organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, 40-100% EtOAc-hexanes gradient elution) afforded 4 as a white solid.

5. Oleic acid (5: FIG. 1)

Oleic acid was obtained from Aldrich chemical company, CAS #112-80-1.

6. Erucamide (6: FIG. 1)

Erucamide was obtained from Aldrich Chemical Company, CAS #28,057-7.

7. Methyl-8-hydroxy-octanoate (7: Scheme 3)

A solution of suberic acid monomethyl ester (1.5 g, 7.97 mmol, 1.0 equiv.) in tetrahydrofuran (THF) (32.0 mL, .25M) at −20° C. was treated dropwise with BH₃.THF (IM solution in THF, 7.97 mL, 7.97 mmol, 1.0 equiv.). The reaction mixture was stirred overnight and was subsequently allowed to reach room temperature. The reaction mixture was then diluted with ethyl acetate (100 mL) and quenched with methanol (10 mL) and 10% HCl (10 mL). Extraction with NaHCO₃ (1×20 mL), water (2×10 mL), and brine (1×10 mL), afforded methyl-8-hydroxy-octanoate

(7) as a crude white solid.

8. Methyl-8-bromo-octanoate (8: Scheme 3)

A solution of crude methyl-8-hydroxy-octanoate (7, 1.24 g, 7.13 mmol, 1.0 equiv.) in CH₂Cl₂ (15 mL, 0.48 M) at 0° C. was treated successively with CBr₄ (3.07 g, 9.27 mmol, 1.3 equiv.) and PPh₃ (2.61 g, 9.98 mmol, 1.4 equiv.) and the reaction mixture was stirred at 4° C. for 10 h. The reaction mixture was then concentrated under reduced pressure and washed repeatedly with Et₂O (8×10 mL washes). The Et₂O washes were combined and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, hexanes) afforded 8 as a clear, colorless oil (1.25 g, 1.69 g theoretical, 74.0%): ¹H NMR (CDCl₃,250 MHz) δ 3.64 (s, 3H, C(O)OCH₃), 3.38 (t, 2H, J=6.8 Hz, CH₂Br), 2.29 (t, 2H, J=7.4 Hz CH₂C(O)OCH₃), 1.83 (p, 2H, CH₂CH₂Br), 1.63 (m, 2H, CH₂CH₂C(O)OCH₃) 1.47-1.28 (m, 6H, alkyl protons).

9. Methyl-8-triphenylphosphoranyl-octanoate-bromide (9: Scheme 3)

A solution of 8 (1.25 g, 5.23 mmol, 1.0 equiv.) in CH₃CN (4.0 mL, 1.31 M) was treated with triphenylphosphine (1.52 g, 5.75 mmol, 1.1 equiv.) and stirred at reflux for 10 h. Additional triphenylphosphine (0.685 g, 2.61 mmol, 0.5 equiv.) was added to the reaction mixture and stirring was continued at reflux for 5 h. The reaction mixture was concentrated under reduced pressure and washed repeatedly with Et₂O (5×10 mL washes). The remaining residue was then solubilized in the minimum volume of CH₂Cl₂ and concentrated under reduced pressure to afford 9 as a colorless foam (2.20 g, 2.61 g theoretical, 84.3%): 1H NMR (CDCl₃,250 MHz) δ 7.82-7.51 (m, 15H, ArH), 3.70-3.46 (m, 5H, CH₃OC(O)R and CH₂PPh₃), 2.13 (t, 2H, J=7.4 Hz, CH₂C(O)OCH₃, 1.62-1.43 (m, 6H, alkyl protons), 1.30-1.02 (m, 4H, alkyl protons); FABHRMS (NBA) m/e 419.2154 (C₂₇H₃₂BrO2P—Br⁻ requires 419.2140).

10. Methyl-cis-8. 9-octadecenoate (10: Scheme 3

A solution of 9 (0.71 g, 1.42 mmol, 1.0 equiv.) in THF (7.0 mL, 0.2 M) at 25° C. was treated with KHMDS (3.0 mL, 0.5 M solution in THF, 1.5 mmol, 1.06 equiv.) and the reaction mixture was stirred at reflux for 1 h. The reaction mixture was then cooled to −78 ° C., treated with decyl aldehyde (0.321 mL, 1.71 mmol, 1.2 equiv.) warmed to 25° C., and stirred for an additional 30 min. The reaction mixture was then treated with saturated aqueous NH₄Cl and partitioned between EtOAc (100 mL) and H₂O (100 mL). The organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, 0-2% EtOAc-hexanes gradient elution) afforded 10 as a colorless oil (0.290 g, 0.422 g theoretical, 68.7 %): ¹H NMR (CDCl₃,250 MHz) δ 5.34 (m, 2H, CH═CH), 3.65 (s, 3H, CH₃OC(O)), 2.29 (t, 2H, J=7.4 Hz, CH₂C(O)OCH₃), 2.00 (m, 4H, CH₂CH═CHCH₂), 1.61 (m, 2H, CH₂CH₂C(O)OCH₃), 1.29 (bs, 20 H, alkyl protons), 0.86 (t, 3H, RCH₃).

11. Cis-8,9octadecenoic acid (11: Scheme 3)

A solution of 10 (0.245 g, 0.825 inmol, 1.0 equiv.) in THF-MeOH-H₂O (3-1-1 ratio, 4.1 mL, 0.2 M) at 0° C. was treated with LiOH.H₂O (0.104 g, 2.48 mrmol, 3.0 equiv.). The reaction mixture was warmed to 25° C., stirred for 8 h, and then partitioned between EtOAc (100 mL) and H₂O (100 mL). The organic layer was washed successively with 10% aqueous HCl (100 mL) and saturated aqueous NaCl (100 mL), dried, and concentrated under reduced pressure. Chromatography (SiO₂,5cm×15 cm, 10-30% EtOAc-hexanes gradient elution) afforded 11 as a colorless oil (0.156 g, 0.233 g theoretical, 67.0%): ¹H NMR (CDCl₃,250 MHz) δ 5.34 (m, 2H, CH═CH), 2.34 (t, 2H, J=7.4 Hz, CH₂COOH), 2.01 (m, 4H, CH₂CH═CHCH₂), 1.61 (m, 2H, CH₂CH₂COOH), 1.47-1.23 (m, 20 H, alkyl protons), 0.87 (t, 3H, RCH₃).

12. 18-Hemisuccinate-cis-9,10-octadecenoamide (12: Scheme 4)

A solution of 18 (0.047 g, 0.160 M, 1.0 equiv) in CH₂Cl₂—CHCl₃ (3-1, 1.60 mL, 0.1M) was treated successively with Et₃N (0.045 mL, 0.320 mmol, 2.0 equiv), succinic anhydride (0.033 g, 0.320 mmol, 2.0 equiv) and DMAP (0.002 g, 0.016 mmol, 0.1 equiv), and the reaction mixture was stirred at 25° C. for 10 h. The reaction mixture was then partitioned between CH₂Cl₂ (50 mL) and H₂O (50 mL), and the organic layer was washed successively with 10% aqueous HCl (50 mL) and saturated aqueous NaCl (50 mL), dried (Na₂SO₄), and concentrated under reduced pressure. Chromatography (SiO₂,3 cm×15 cm, 0-10% MeOH-EtOAc) afforded 12 as a white solid (0.051 g, 0.063 theoretical, 80.3%): ¹H NMR (CDCl₃,250 MHz) δ 6.95 (b s, 1H, H₂NC(O)), 5.72 (b s, 1H, H₂NC(O)), 5.34 (m, 2H, CH═CH), 4.08 (t, 3H, J=6.6 Hz, CH₂OC(O)R), 2.61 (m, 4H, ROC(O)CH₂CH₂COOH), 2.21 (t, 2H, J=7.5 Hz, CH₂C(O)NH₂), 2.00 (m, 4H, CH₂CH═CHCH₂), 1.70-1.52 (m, 4H, CH2CH₂C(O)NH₂ and CH₂CH₂OH), 1.29 (b s, 18H, alkyl protons); FABHRMS (NBA) m/e 398.2893 (C₂₂H₃₉NO₅ +H+requires 398.2906).

13. Methyl-9-bromo-nonanoate (13: Scheme 4)

A solution of methyl-9-hydroxy-nonanoate (1.1 g, 5.85 mmol, 1.0 equiv) in CH₂Cl₂ (30 mL, 0.2 M) at 0° C. was treated successively with CBr₄ (2.5 g, 7.54 mmol, 1.3 equiv) and PPh₃ (2.15 g, 8.19 mmol, 1.4 equiv) and the reaction mixture was stirred at 4° C. for 10 h. The reaction mixture was then concenctrated under reduced pressure and washed repeatedly with Et₂O (8×10 mL washes). The Et₂o washes were combined and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, hexanes) afforded 13 as a clear, colorless oil (1.02 g, 1.47 g theorectical, 69.5 %): ¹H NMR (CDCl₃,250 MHz) d 3.64 (s, 3H, C(O)OCH₃), 3.38 (t, 2H, J=6.8 Hz, CH₂Br), 2.29 (t, 2H, J=7.4 Hz CH₂C(O)OCH₃), 1.83 (p, 2H, CH₂CH₂Br), 1.63 (m, 2H, CH₂CH₂C(O)OCH₃) 1.47-1.28 (m, 8H, alkyl protons).

14. Methyl-9-triphenylphosphoranyl-nonanoate-bromide (14: Scheme 4)

A solution of 13 (1.02 g, 4.06 mmol, 1.0 equiv) in CH₃CN (3.5 mL, 1.16 M) was treated with triphenylphosphine (1.17 g, 4.47 mmol, 1.1 equiv) and stirred at reflux for 10 h. Additional triphenylphosphine (0.532 g, 2.03 mmol, 0.5 equiv) was added to the reaction mixture and stirring was continued at reflux for 5 h. The reaction mixture was concentrated under reduced pressure and washed repeatedly with Et₂O (5×10 mL washes). The remaining residue was then solubilized in the minimum volume of CH₂Cl₂ and concentrated under reduced pressure to afford 14 as a colorless foam (1.90 g, 2.08 g theoretical, 91.3%): ¹H NMR (CDCl₃,250 MHz) δ 7.82-7.51 (m, 15H, ArH), 3.70-3.46 (m, 5H, CH₃OC(O)R and CH₂PPh₃), 2.13 (t, 2H, J=7.4 Hz, —CH₂C(O)OCH₃), 1.62-1.02 (m, 12H, alkyl protons); FABHRMS (NBA) m/e 433.2312 (C₂₈H₃₄BrO₂P—Br⁻ requires 433.2296).

15. Methyl-18-t-butyldiphenysilyloxy-cis-9,10 octadecenoate (15: Scheme 4)

A solution of 14 (1.0 g, 1.95 mmol, 1.0 equiv) in THF (6.5 mL, 0.3 M) at 25° C. was treated with KHMDS (3.9 mL, 0.5 M solution in THF, 1.95 mmol, 1.0 equiv) and the reaction mixture was stirred at reflux for 1 h. The reaction mixture was then cooled to −78° C., treated with 3 (0.93 g, 2.35 mmol, 1.2 equiv), warmed to 25° C., and stirred for an additional 30 min. The reaction mixture was then treated with saturated aqueous NH₄C1 and partitioned between EtOAc (100 mL) and H₂O (100 mL). The organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, 0-2% EtOAc-hexanes gradient elution) afforded 15 as a colorless oil (0.82 g, 1.07 g theoretical, 76.3%): ¹H NMR (CDCl₃,250 MHz) δ 7.67 (m, 4H, ArH), 7.41 (m, 6H, ArH), 5.34 (m, 2H, CH═CH), 3.65 (m, 5H, CH₃OC(O) and CH₂OTBDPS), 2.29 (t, 2H, J=7.4 Hz, CH₂C(O)OCH3), 2.00 (m, 4H, CH₂CH═CHCH₂), 1.55 (m, 4H, CH₂CH₂C(O)OCH₃ and CH₂CH₂OTBDPS), 1.29 (b s, 18H, alkyl protons), 1.04 (s, 9H, (CH₃)₃C).

16. 18-T-butyldiphenylsilyloxy-cis-9,10-octadecenoic acid (16: Scheme 4)

A solution of 5 (0.81 g, 1.47 mmol, 1.0 equiv) in THF-MeOH-H₂O (3-1-1 ratio, 7.3 mL, 0.2 M) at 0° C. was treated with LiOH.H₂O (0.188 g, 4.48 mmol, 3.0 equiv). The reaction mixture was warmed to 25° C., stirred for 8 h, and then partitioned between EtOAc (100 mL) and H₂O (100 mL). The organic layer was washed successively with 10% aqueous HCl (100 mL) and saturated aqueous NaCl (100 mL), dried, and concentrated under reduced pressure. Chromatography (SiO₂, 5 cm×15 cm, 10-30% EtOAc-hexanes gradient elution) afforded 16 as a colorless oil (0.700 g, 0.790 g theoretical, 88.7%): ¹H NMR (CDCl₃, 250 MHz) δ 7.67 (m, 4H, ArH), 7.41 (m, 6H, ArH), 5.34 (m, 2H, CH═CH), 3.65 (t, 3H, J=6.5 Hz, CH₂OTBDPS), 2.34 (t, 2H, J=7.4 Hz, CH₂COOH), 2.00 (m, 4H, CH₂CH═CHCH₂), 1.65-1.50 (m, 4H, CH₂CH₂COOH and CH₂CH₂OTBDPS), 1.47-1.23 (m, 18H, alkyl protons), 1.05 (s, 9H, (CH3)₃C); FABHRMS (NBA/CsI) m/e 669.2772 (C₃₄H₅₂O₃Si+Cs⁺ requires 669.2740).

17. 18-T-butyldiphenylsilyloxy-cis-9,10-octadecenoamide (17: Scheme 4)

A solution of 16 (0.685 g, 1.28 mmol, 1.0 equiv) in CH₂Cl₂ (4.3 mL, 0.3 M) at 0° C. was treated dropwise with oxalyl chloride (1.92 mL, 2 M solution in CH₂Cl₂,3.84 mmol, 3.0 equiv). The reaction mixture was stirred at 25° C. for 4 h, concentrated under reduced pressure, cooled to 0° C., and treated with saturated aqueous NH₄OH (2.0 mL). The reaction mixture was then partitioned between EtOAc (100 mL) and H₂O (100 mL), and the organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, 40-100% EtOAc-hexanes gradient elution) afforded 17 as a colorless oil (0.520 g, 0.684 g, 76.0%): ¹H NMR (CDCl₃,250 MHz) δ 7.67 (m, 4H, ArH), 7.41 (m, 6H, ArH), 5.70-5.34 (m, 4H, H₂NC(O) and CH═CH), 3.65 (t, 3H, J=6.5 Hz, CH₂OTBDPS), 2.21 (t, 2H, J=7.5 Hz, CH₂C(O)NH₂), 2.00 (m, 4H, CH₂CH═CHCH₂), 1.65-1.50 (m, 4H, CH₂CH₂C(O)NH₂ and CH₂CH₂OTBDPS), 1.47-1.23 (m, 18H, alkyl protons), 1.05 (s, 9H, (CH₃)₃C); FABHRMS (NBA/CsI m/e 668.2929 (C₃₄H₅₃O₂NSi+Cs⁺ requires 668.2900).

18. 18-Hydroxy-cis-9,10-octadecenoamide (18: Scheme 4)

A solution of 17 (0.185 g, 0.345 mmol, 1.0 equiv) in THF (1.1 mL, 0.31 M) was treated with tetrabutylammoniumfluoride (0.69 mL, 1.0 M solution in THF, 0.69 mmol, 2.0 equiv) and the reaction mixture was stirred at 25° C. for 2 h. The reaction mixture was then partitioned between EtOAc (50 mL) and H₂O (50 mL), and the organic layer was dried (Na₂SO₄) and concentrated under reduced pressure . Chromatography (SiO₂,3 cm×15 cm, 0-5% MeOH-EtOAc gradient elution) afforded 18 as a white solid (0.097 g, 0.103 g theoretical, 94.6%): ¹H NMR (CDCl₃,250 MHz) δ 5.65-5.34 (m, 4H, H₂NC(O) and CH═CH), 3.62 (t, 3H, J=6.5 Hz, CH₂OH), 2.21 (t, 2H, J=7.5 Hz, CH₂C(O)NH₂), 2.00 (m, 4H, CH₂CH═CHCH₂), 1.65-1.50 (m, 4H, CH₂CH₂C(O)NH₂ and CH₂CH₂0H), 1.29 (b s, 18H, alkyl protons); FABHRMS (NBA) 298.2732 (C₁₈H₃₅NO₂+H⁺ requires 298.2746).

19. Synthesis of Compound 100 (FIG. 5)

Methyl-9-t-butyldiphenylsilyloxy-nonanoate (intermediate for compound 100: FIG. 5). A solution of methyl-9-hydroxy-nonanoate (0.838 g, 4.46 mmol, 1.0 equiv: Aldrich) in CH₂Cl₂ (15 mL, 0.3 M) was treated successively with Et₃N (0.75 mL, 5.38 mmol, 1.2 equiv), t-butylchlorodiphenylsilane (1.28 mL, 4.93 mmol, 1.1 equiv), and DMAP (0.180 g, 1.48 mmol, 0.33 equiv), and the reaction mixture was stirred at 25° C. for 12 h. Saturated aqueous NH₄Cl was added to the reaction mixture and the mixture was partitioned between CH₂Cl₂ (100 mL) and H₂O (100 mL). The organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Chromatography (SiO₂, 5 cm×15 cm, 0-5% EtOAc-hexanes gradient elution ) afforded the intermeidate as a clear, colorless oil (1.22g, 1.831 theoretical, 64.1%): ¹H NMR (CDCl₃,250 MHz) δ 7.66 (m, 4H, ArH), 7.38 (m, 6H, ArH), 3.67-3.62 (m, 5H, C(O)OCH₃ and CH₂OTBDPS), 2.30 (t, 2H, J=7.4 Hz, CH₂C(O)OCH₃), 1.58 (m, 4H, CH₂CH₂OTBDPS and CH₂CH₂C(O)OCH₃), 1.28 (b s, 8H, alkyl protons), 1.05 (s, 9H, C(CH₃) ₃)

20. Methyl-9-bromo-nonanoate (Intermediate for Compound 100: FIG. 5)

A solution of methyl-9-hydroxy-nonanoate (1.1 g, 5.85 mmol, 1.0 equiv) in CH₂Cl₂ (30 mL, 0.2 M) at 0° C. was treated successively with CBr₄ (2.5 g, 7.54 mmol, 1.3 equiv) and PPh₃ (2.15 g, 8.19 mmol, 1.4 equiv) and the reaction mixture was stirred at 4° C. for 10 h. The reaction mixture was then concenctrated under reduced pressure and washed repeatedly with Et₂O (8×10 mL washes). The Et₂O washes were combined and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, hexanes) afforded the intermediate as a clear, colorless oil (1.02 g, 1.47 g theorectical, 69.5 %): ¹H NMR (CDCl₃,250 MHz) d 3.64 (s, 3H, C(O)OCH₃), 3.38 (t, 2H, J=6.8 Hz, CH₂Br), 2.29 (t, 2H, J=7.4 Hz CH₂C(O)OCH_(3), 1.83 (p, 2H, c)H₂CH₂Br), 1.63 (m, 2H, CH₂CH₂C(O)OCH₃) 1.47-1.28 (m, 8H, alkyl protons).

21. 9-T-butyldiphenylsilyloxy-nonanal (Intermediate for Compound 100: FIG. 5)

A solution of 1 (1.25 g, 2.93 mmol, 1.0 equiv) in toluene (9.80 mL, 3.0 N) at −78° C. was treated dropwise with DIBAL-H (4.40 mL, 1.0 M solution in hexanes, 4.40 mmol, 1.5 equiv). The reaction mixture was stirred at −78° C. for 30 min. The reaction mixture was then treated dropwise with MeOH (2 mL) and partitioned between EtOAc (100 mL) and H₂O (100 mL). The organic layer was washed with 10 % aqueous HCl (100 mL), dried (Na₂SO₄), and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, 0-5 % EtOAc-hexanes gradient elution) afforded 3 as a colorless oil (1.1 g, 94.9 %): ¹H NMR (CDCl₃,250 MHz) 6 9.76 (t, 1H, J=1.8 Hz, HC(O)R), 7.67 (m, 4H, ArH), 7.40 (m, 6H, ArH), 3.65 (t, 2H, J=6.4 Hz, CH₂OTBDPS), 2.41 (t of d, 2H J=1.8 and 7.3 Hz, CH₂C(O)H), 1.58 (m, 4H, CH₂CH₂OTBDPS and CH₂CH₂C(O)H), 1.29 (b s, 8H, alkyl protons), 1.05 (s, 9H, (CH₃)₃C); FABHRMS (NBA/CsI) m/e 529.1560 (C25H36O2Si+Cs⁺ requires 529.1539).

22. Methyl-9-triphenylphosphoranyl-nonanoate Bromide (Intermediate for Compound 100: FIG. 5)

A solution of 9-T-butyldiphenylsilyloxy-nonanal (1.02 g, 4.06 mmol, 1.0 equiv) in CH₃CN (3.5 mL, 1.16 M) was treated with triphenylphosphine (1.17 g, 4.47 mmol, 1.1 equiv) and stirred at reflux for 10 h. Additional triphenylphosphine (0.532 g, 2.03 mmol, 0.5 equiv) was added to the reaction mixture and stirring was continued at reflux for 5 h. The reaction mixture was concentrated under reduced pressure and washed repeatedly with Et₂O (5×10 mL washes). The remaining residue was then solubilized in the minimum volume of CH₂Cl₂ and concentrated under reduced pressure to afford the intermediate as a colorless foam (1.90 g, 2.08 g theoretical, 91.3%): ¹H NMR (CDCl₃,250 MHz) δ 7.82-7.51 (m, 15H, ArH), 3.70-3.46 (m, 5H, CH₃OC(O)R and CH₂PPh₃), 2.13 (t, 2H, J=7.4 Hz, CH₂C(O)OCH₃), 1.62-1.02 (m, 12H, alkyl protons); FABHRMS (NBA) m/e 433.2312 (C ₂₈H₃₄BrO₂P—Br⁻ requires 433.2296).

23. Methyl-18-t-butyldiphenysilyloxy-cis-9,10-octadecenoate (Intermediate for Compound 100: FIG. 5)

A solution of (1.0 g, 1.95 mmol, 1.0 eguiv) in THF (6.5 mL, 0.3 M) at 25 ° C. was treated with KHMDS (3.9 mL, 0.5 M solution in THF, 1.95 mmol, 1.0 equiv) and the reaction mixture was stirred at reflux for 1 h. The reaction mixture was then cooled to −78 ° C., treated with 3 (0.93 g, 2.35 mmol, 1.2 equiv), warmed to 25° C., and stirred for an additional 30 min.

The reaction mixture was then treated with saturated aqueous NH₄Cl and partitioned between EtOAc (100 mL) and H₂O (100 mL). The organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Chromatography (Sio₂,5 cm×15 cm, 0-2% EtOAc-hexanes gradient elution) afforded the intermediate as a colorless oil (0.82 g, 1.07 g theoretical, 76.3%): ¹H NMR (CDCl₃,250 MHz) δ 7.67 (m, 4H, ArH), 7.41 (m, 6H, ArH), 5.34 (m, 2H, CH═CH), 3.65 (m, 5H, CH₃OC(O) and CH₂OTBDPS), 2.29 (t, 2H, J=7.4 Hz, CH₂C(O)OCH₃), 2.00 (m, 4H, CH₂CH═CHCH₂), 1.55 (m, 4H, CH₂CH₂C(O)OCH₃ and CH₂CH₂OTBDPS), 1.29 (b s, 18H, alkyl protons), 1.04 (s, 9H, (CH₃)₃C).

24. 18-T-butyldiphenylsilyloxy-cis-9,10-octadecenoic acid (Compound 100: FIG. 5)

A solution of Methyl-18-t-butyldiphenysilyloxy-cis-9,10-octadecenoate (0.81 g, 1.47 mmol, 1.0 equiv) in THF-MeOH-H₂O (3-1-1 ratio, 7.3 mL, 0.2 M) at 0° C. was treated with LiOH.H₂O (0.188 g, 4.48 mmol, 3.0 equiv). The reaction mixture was warmed to 25° C., stirred for 8 h, and then partitioned between EtOAc (100 mL) and H₂O (100 mL). The organic layer was washed successively with 10% aqueous HCl (100 mL) and saturated aqueous NaCl (100 mL), dried, and concentrated under reduced pressure. Chromatography (SiO₂,5 cm×15 cm, 10-30% EtOAc-hexanes gradient elution) afforded 100 as a colorless oil (0.700 g, 0.790 g theoretical, 88.7%): ¹H NMR (CDCl₃,250 MHz) δ 7.67 (m, 4H, ArH), 7.41 (m, 6H, ArH), 5.34 (m, 2H, CH═CH), 3.65 (t, 3H, J=6.5 Hz, CH₂OTBDPS), 2.34 (t, 2H, J=7.4 Hz, CH₂COOH), 2.00 (m, 4H, CH₂CH═CHCH₂), 1.65-1.50 (m, 4H, CH₂CH₂COOH and CH₂CH₂OTBDPS), 1.47-1.23 (m, 18H, alkyl protons), 1.05 (s, 9H, (CH₃)₃C); FABHRMS (NBA/CsI) m/e 669.2772 (C₃₄H₅₂O₃Si+Cs⁺ requires 669.2740). 25. Synthesis of Compound 101 (FIG. 5)

Step 1.

A solution of 100 (1.0 equiv) in CH₂Cl₂ (0.3 M) at 0° C. was treated dropwise with oxalyl chloride (4.0 equiv). The reaction mixture was stirred at 25° C. for 4 h, concentrated under reduced pressure, cooled to 0° C., and treated with saturated aqueous NH₄OH (2.0 mL). The reaction mixture was then partitioned between EtOAc (100 mL) and H₂O (100 mL), and the organic layer was dried (Na₂SO₄) and concentrated under reduced pressure.

Step 2.

A solution of the above step 1 intermediate compound (1.0 equiv) in ether (0.3 M) at 0° C. was treated dropwise with pyridine (8.0 equiv.) followed by trifluoroaceticanhydride (6.0 equiv; Aldrich). The reaction mixture was stirred at 25° C. for 3 h, concentrated under reduced pressure, cooled to 0° C., and treated with saturated aqueous NH₄OH (2.0 mL). The reaction mixture was then partitioned between EtOAc (100 mL) and H₂O (100 mL), and the organic layer was dried (Na₂SO₄) and concentrated under reduced pressure.

Step 3.

A solution of the above step 2 intermediate compound (1.0 equiv) in THF (0.31 M) was treated with tetrabutylammoniumfluoride (1.0 M solution in THF, 3.0 equiv) and the reaction mixture was stirred at 25° C. for 3 h. The reaction mixture was then partitioned between EtOAc (50 mL) and H₂O (50 mL), and the organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. Product was purified by standard chromatographic conditions and yielded compound 101 in 66% overall yield for the 3 steps.

26. Synthesis of Compound 102 (FIG. 5)

Step 1.

A solution of 101 (1.0 equiv.) in THF (0.1 M) was treated with triphenylphosphine (2.0 equiv.), followed by diethylazodicarboxylate solution (1.0 THF solution, DEAD, 2.0 equiv., Aldrich) and at 0° C. for 30 minutes. The reaction mixture was concentrated under reduced pressure and washed repeatedly with Et₂O (5×10 mL washes). The remaining residue was then solubilized in the minimum volume of CH₂Cl₂ and concentrated under reduced pressure.

Step 2.

A solution of the above step 1 compound (1.0 equiv.) in THF (0.10 M) was treated with thiolacetic acid (2.0 equiv.; Aldrich) at 0° C. for 30 minutes. The reaction mixture was concentrated under reduced pressure and washed repeatedly with Et₂O (5×10 mL washes). The remaining residue was then solubilized in the minimum volume of CH₂Cl₂ and concentrated under reduced pressure. Product was purified by standard chromatographic conditions and yielded compound 102 in 71% overall yield for the 2 steps.

27. Synthesis of Compound 103 (FIGS. 4 & 5)

Step 1.

A solution of 102 (1.0 equiv) in MeOH/Water (2:1 mixture, total concentration 0.20 M) at 0° C. was treated with NaOH (3.0 equiv) and stirred for 10 minutes, and then partitioned between EtOAc (100 mL) and water (100 mL). The organic layer was washed successively with 10% aqueous HCl (100 mL) and saturated aqueous NaCl (100 mL), dried, and concentrated under reduced pressure.

Step 2.

A solution of the above step 1 compound (1.0 equiv) in aqueous 1N HCl at 0° C. was stirred until the reaction mixuture achieved a pH of 7.0, and then the mixture was partitioned between EtOAc (100 mL) and water (100 mL). The organic layer was washed successively with saturated aqueous NaCl (100 mL), dried, and concentrated under reduced pressure.

Step 3. A solution of the above step 2 compound (1.0 equiv.) in aqueos 1 mM NaHCO₃ at 25° C. was treated with Pyridyl disulfide beads (1.1 equiv. Aldrich) and stirred for 2 hours. The beads were subsequently washed with excess saturated NaHCO₃(3×), water (3×) and brine (1×). Standard filtration obtained the activated beads (compound 103) which were then packed into the column for affinity chromatography of the enzyme as discussed supra using this CF3-inhibitor linked to activated pyridyl disulphide beads.

D. Cloning of Cis-9,10-Octadecenoamidase cDNA

1. Cis-9,10-Octadecenoamidase cDNA Obtained from Rat Liver mRNA

To obtain a CDNA clone for cis-9,10-octadecenoamidase from CDNA library generated from rat liver mRNA, degenerate oligonucleotide primers were designed based on the amino acid residue sequence of cis-9,10-octadecenoamidase polypeptide fragment obtained from a trypsin digest. Briefly, the cis-9,10-octadecenoamidase, purified as described above, was subjected to a trypsin digest to form internal polypeptide fragments as performed by Worchester Foundation, Worchester, Pa. The resultant polypeptide fragments were purified by HPLC and seven HPLC fractions showing discrete peptide masses as measured by Matrix-Assisted-Laser-Desorption-Ionization with Time-of-Flight (MALDI TOF, PerSeptive Biosystems Linear Instrument) mass spectrometry were selected for microsequencing. Seven polypeptide fragments were microsequenced having lengths ranging from 12 to 25 amino acid residues as indicated in FIG. 9 indicated by seven discontinuous singly underlined regions in the complete rat cis-9,10-octadecenoamidase amino acid residue sequence. Each peptide possessed the required lysine or arginine residue at its C-terminus indicating that the tryptic digest proceeded with the anticipated selectivity.

The degenerate oligonucleotide primers were designed to incorporate a unique restriction site into the 5′ ends of the primers that functioned as either forward and the backward primers. The forward primers are also referred to as upstream, sense or 5′ primers. The backward primers are also referred to as downstream, anti-sense or 3′ primers. The restriction sites were incorporated into the polymerase chain reaction (PCR) products to allow for insertion into the multiple cloning site of a sequencing vector as described below.

The synthesized 5′ and 3′ degenerate oligonucleotides were designed respectively corresponding to portions of sequenced peptides 1 and 2 as shown in FIG. 9 as indicated by the first two discontinuous singly underlined amino acid residue sequences. The degenerate nucleotides are indicated by IUPAC codes N=A, C, G or T and R =A or G. The nucleotide sequence of the 5′ degenerate primer corresponding to peptide 1 was 5′ CGGAATTCGGNGGNGARGGNGC3′ (SEQ ID NO 3) incorporating an EcoRI restriction site and translating into the amino acid sequence GGEGA (SEQ ID NO 4). The nucleotide sequence of the 3′ degenerate primer that corresponded to peptide 2 was 5′ CGGGATCCGGCATNGTRTARTTRTC3′ (SEQ ID NO 33) incorporating an BamHI restriction site and translating into the amino acid sequence DNYTMP (SEQ ID NO 34).

To amplify regions of cDNA encoding cis-9,10-octadecenoamidase, rat liver mRNA was reversed transcribed into cDNA for use a template in PCR with selected pairs of degenerate oligonucleotide primers described above. PCR was performed under conditions well known to one of ordinary skill in the art with each cycle of 40 total cycles having the temperatures 94° C. for 30 seconds, 60° C. for 45 seconds and 72° C. for 60 seconds.

Of the cloned PCR fragments, three were selected for sequencing. The three PCR fragments were 350 base pairs (bp), 400 bp and 750 bp. Sequencing of these cis-9,10-octadecenoamidase-encoding cDNA fragments showed that the 750 bp fragment contained the sequences of both the 350 and 400 bp fragments.

The 350 bp cDNA fragment obtained by PCR was then labeled internally and used as a probe for Northern analysis on electrophoresed rat liver mRNA. The probe hybridized to a fragment approximately 2.5 to 3.0 kilobases (kb) in length, which is the expected size of the cis-9,10-octadecenoamidase mRNA that encodes a 60 kDa protein.

To isolate a cDNA clone encoding the complete cis-9,10-octadecenoamidase protein, the 350 bp probe was then internally labeled with ³²P used to screen a λgtll cDNA library from rat liver mRNA obtained from Clontech (Palo Alto, Calif.). For screening, the amplified 350 bp fragment was first digested with EcoRI and BamHI for directional cloning into a similarly digested pBluescript II SK(−)(Stratagene, La Jolla, CA). The resultant sequence indicated that the 350 bp fragment encoded the peptides 1 and 2 from which the degenerate oligonucleotide primers were designed confirming the accuracy of the PCR and amplification of the desired clone. The methods for cloning the cis-9,10-octadecenoamidase cDNA of this invention are techniques well known to one of ordinary skill in the art and are described, for example, in “Current Protocols in Molecular Biology”, eds. Ausebel et al., Wiley & Sons, Inc., New York (1989), the disclosures of which are hereby incorporated by reference.

Four positive clones were identified from a screening of 4.5×10⁵ plaques. Two clones of 2.7 kb in length and 1 of 2.0 kb in length, were obtained. The partial sequence of one of the 2.7 kb clones, designated p60, indicates that the clone does contain cis-9,10-octadecenoamidase-specific sequences.

The rat liver cDNA clone designated p60 obtained above has been deposited with American Type Culture Collection (ATCC) on or before Jun. 12, 1996 and has been assigned the ATCC accession number 97605. This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable plasmid for 30 years from the date of each deposit. The plasmid will be made available by ATCC under the terms of the Budapest Treaty which assures permanent and unrestricted availability of the progeny of the plasmid to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638). The assignee of the present application has agreed that if the plasmid deposit should die or be lost or destroyed when cultivated under suitable conditions, it will be promptly replaced on notification with a viable specimen of the same plasmid. Availability of the deposit is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

A partial nucleotide sequence of the top strand of the p60 cDNA clone containing 780 nucleotides described above is listed in SEQ ID NO 1 along with the deduced amino acid residue sequence. The encoded amino acid residue sequence is listed separately in SEQ ID NO 2. In order to show the amino acid residue encoded by each triplet codon in the Sequence Listing, a stop codon, TAA, was added at positions 781 to 783 to allow for the coding sequence (CDS) function in the Patentin program used to prepare the Sequence Listing. In other words, the stop codon is artificially inserted into the nucleotide sequence shown in SEQ ID NO 1 to facilitate the translation of the cDNA coding sequence into an amino acid sequence.

The actual position of the cis-9,10-octadecenoamidase nucleotide position within a complete cDNA clone is evident from the complete cDNA sequence as described below.

The two largest positive cDNA clones were then cloned into pBluescript II SK(+) and sequenced. One clone encoded a partially processed transcript containing the full coding sequence of the oleamide amidase with an additional 200 bp of intronic sequence. The other clone encoded a fully processed oleamide amidase transcript but fused to the 5′ end of the clone was a 300 bp fragment encoding rRNA. Fusion of the two clones through an internal overlapping HindIII restriction site generated the full-length rat cis-9,10-octadecenoamidase also referred to as fatty acid amide hydrolase abbreviated as FAAH. The clone was sequenced with sequencing primers that were synthesized on a Beckman Oligo1000M Synthesizer.

The resultant full length rat cDNA FAAH clone, also referred to as rFAAH cDNA, contained 2473 bp, which contained a single 1.73 kb open reading frame encoding 63.3 kDa of protein sequence as shown in FIGS. 10-1 to 10-5. The double-stranded rat FAAH cDNA sequence is available by GenBank with Accession Number U72497. The encoded rat FAAH protein is also referred to as rFAAH protein. The clone contained 50 bp of sequence 5′ to the first ATG designation the start of the open reading frame. The clone also contained 685 bp of 3′ untranslated region between the first stop codon indicating the end of the open reading frame and the poly A tail.

In FIGS. 10-1 through 10-5, the encoded amino acid residue is positioned directly underneath the second nucleotide of a triplet codon. For example, at the initiation site where ATG encodes methionine (M), the A nucleotide begins at nucleotide position 50 and the G nucleotide is 52. The encoded M is located underneath the T nucleotide at nucleotide position 51. As presented in the figure, thus, the indicated triplet codons are not as indicated. The top and bottom strands of the cDNA sequence are also respectively listed as SEQ ID NOs 35 and 37. The encoded amino acid sequence is shown in with the top strand in SEQ ID NO 35 and again by itself in SEQ ID NO 36.

Although the 50 bases of nucleotide sequence upstream of the first ATG did not possess an in-frame stop codon, the following several lines of evidence supported the 2.47 kb CDNA encoding the complete oleamide hydrolase protein sequence: 1) The size of the cDNA matched closely the predicted size of the mRNA transcript as estimated by Northern blot (FIG. 12B as discussed below); 2) The sequence surrounding the first ATG possessed the required consensus sequence for eukaryotic translation initiation sites, in particular, an A is present at the −3 position and a G is present at the +4 position; and 3) When transiently transfected with oleamide hydrolase cDNA, COS-7 cells translated a functional protein product that comigrated with affinity isolated oleamide hydrolase on SDS-PAGE (FIG. 12B, lane 1 and discussed below).

Database searches with the oleamide amidase protein sequence (FAAH) identified strong homology to several amidase enzyme sequences from organisms as divergent as Agrobacterium tumefaciens (Klee et al., Proc. Nat . Acad. Sci., USA, 81:1728-1732, 1984), Pseudomonas savastanoi (Yamada et al., Proc. Natl. Acad. Sci., USA, 82:6522-6526, 1985); Aspergillus nidulans (Corrick et al., Gene, 53:63-71, 1987), Saccharomyces cerevisiae (Chang et al., Nuc. Acids Res., 18:7180, 1990), Caenorhabditis elegans (Wilson et al., Nature, 368:32-38, 1994), and Gallus domesticus (Ettinger et al., Arch. Biochem. Biophys., 316:14-19, 1995). These amidases collectively compose a recently defined enzyme family (Mayaux et al., J. Bacteriol., 172:6764-6773, 1990) whose members all share a common signature sequence as shown in FIG. 11. The encoded amino acids beginning at position 215 and extending through 246 of the rat fatty acid amide hydrolase (oleamide hydrolase or FAAH) contain residues that are found in a family of D M amidases. The sequence in the cis-9,10-octadecenoamidase rat protein of this invention has is shown in SEQ ID NO 36 at amino acid positions 215 to 246. The alignment over the amidase signature sequence region of the rat FAAH with several other representative amidases reveals that the signature sequence is completely conserved among the amidase family members. Those amino acids are shown in bold faced type in the figure and the relative amino acid position of the signature sequence in each amidase is given by the numbers just preceding and following the sequence information. The assigned SEQ ID NOs for each of the sequences are listed in the legend to the Figure in Brief Description of the Figures.

To our knowledge an, oleamide amidase also referred to as FAAH is the first mammalian member of this enzyme family to have been molecularly characterized.

Hydropathicity plot and transmembrane domain searches (TMpred and PSORT programs) of the rat FAAH sequence were conducted, and each search indicated a strong putative transmembrane domain from amino acids 13-29 (bold type in FIG. 9). The 50 amino acid region surrounding and encompassing the putative transmembrane domain of rat FAAH shares no homology with protein sequences of other amidase family members, indicating that one of the unique modifications of the rat amidase may be its integration into the membrane. Interestingly, additional analysis of the FAAH sequence revealed a polyproline segment, amino acids 307-315 (double underlined in FIG. 9), that contains a precise match from positions 310 to 315 to the consensus class II SH3 domain binding sequence, PPLPXR (SEQ ID NO 38) Feng et al, Science, 266:1241-1246, 1994), suggesting that other proteins may interact with FAAH to regulate its activity (Pawson, Nature, 373:573-580, 1995) and/or subcellular localization (Rotin et al, EMBO J., 13:4440-4450, 1994).

Southern and Northern blot analyses were conducted with an internal 800 bp fragment of the rat FAAH CDNA to evaluate the genomic copy number and tissue distribution of FAAH, respectively.

For the Southern blot, 10 Mg of rat genomic DNA was digested with the indicated restriction enzymes (100 units each) for 12 hours and then run on a 0.8% agarose gel. Rat genomic DNA was first isolated from rat liver as follows: approximately 500 mg of rat liver was shaken overnight at 55° C. in 2 ml of 100 mM Tris (pH 8.0), 0.2% SDS, 200 mM NaCl, and 0.2 mg/ml of proteinase K. The mixture was then spun at 15,000 rpm for 15 minutes and the supernatant was removed and treated with an equal volume of isopropanol. The precipitated genomic DNA was removed, partially dried, and resuspended in water by heating at 55° C. for 4 hours. 10 Mg of the DNA was digested with the indicated restriction enzymes (100 units each) for 12 hours, and then run on a 0.8% agarose gel. The DNA was then transferred under capillary pressure to a GeneScreenPlus hybridization transfer membrane (DuPont NEN) for use in Southern blot analysis. The blot was handled according to manufacturer's (Clontech) guidelines and subjected to the following post-hybridization washes: one 20 minute wash in a solution of 1% SDS and 0.2×SSC (30 mM NaCl, 3.0 mM sodium citrate, pH 7.0) at 25° C., followed by two 20 minute washes in a solution of 0.1% SDS and 0.2×SSC at 65° C. and one additional post-hybridization wash (0.1% SDS, 0.1×SSC, pH 7.0) at 65° C. for 1 hour. The blot was then exposed to X-ray film for 12 hours at −78° C.

Southern blot studies showed that the FAAH probe hybridized primarily to single DNA fragments using several different restriction digests of the rat genome (FIG. 12A). As expected, two hybridizing bands were observed in the HindIII digested DNA, as the FAAH probe contained an internal HindIII site. These results are most consistent with the FAAH gene being a single copy gene.

For Northern analyses, blots obtained from Clontech were handled according to manufacturer's guidelines, except that an additional post-hybridization wash with a solution of 0.1% SDS and 0.1×SSC (15 mM NaCl, 1.5 mM sodium citrate, pH 7.0) at 65° C. for 1 hour was conducted to ensure removal of nonspecific hybridization. The resulting blot was exposed to X-ray film for 6 hours at −78° C.

Northern blot analysis with the FAAH probe identified a single major mRNA transcript of approximately 2.5 kb in size that is most abundant in liver and brain, with lesser amounts present in spleen, lung, kidney, and testes (FIG. 12B). This transcript was not detectable in either heart or skeletal muscle, consistent with previously reported biochemical studies identifying no anandamide hydrolase activity in these two tissues (Deutsch et al, Biochem. Pharmacol., 46:791-796, 1993). The Northern blot also contained low level hybridization of the FAAH cDNA probe to a few larger transcripts present only in those tissues expressing the 2.5 kb transcript as well. These transcripts may be either unprocessed or alternatively spliced forms of the 2.5 kb mRNA. In addition, the regional distribution of the rat FAAH transcript in the rat brain was examined by Northern analysis revealing highest level of the hippocampus and thalamus with lower levels of transcript detectable in other regions of the brain, including olfactory bulb, cortex, cerebellum and pituitary. Preliminary in situ hybridization analysis of rat brain slices has also identified high expression levels for rat FAAH in both hippocampus and hypothalamus. Lastly, Northern analysis of mouse FAAH expression levels at various stages in mouse embryonic development was performed where the mouse FAAH was first observed between days 11 and 15 with levels continuing to increase dramatically from day 15 to 17.

2. Cis-9,10-Octadecenoamidase cDNA Obtained from Mouse Liver MRNA

The mouse homolog of the rat cis-9,10-Octadecenoamidase cDNA was obtained from screening a mouse liver 5′-stretch plus cDNA library (Clontech) using the same conditions as described above for obtaining the rat cDNA with the one exception that the entire rat cDNA (FIG. 10-1 through 10-5) was used as the labeled probe.

The resultant mouse double-stranded 1959 bp cDNA homolog and encoded amino acid residue is shown in FIG. 13-1 through 13-4 with the ATG start site beginning at nucleotide position 7 indicated with the boxed methionine (M) residue. The stop codon, TGA, is similarly boxed as shown on FIG. 13-4 at nucleotide positions 1744 to 1746 followed by the 3′ untranslated region. The top and bottom strands of the cDNA sequence are also respectively listed as SEQ ID Nos 39 and 41. The encoded amino acid sequence is shown in with the top strand in SEQ ID NO 39 and again by itself in SEQ ID NO 40.

3. Cis-9,10-Octadecenoamidase CDNA Obtained from Human Liver mRNA

A cDNA clone for the human homolog of cis-9,10-octadecenoamidase was similarly obtained as described above for the rat by screening a human liver 5′ stretch plus cDNA library (Clontech) with the exception that the entire rat cDNA prepared above was used as the labeled probed and less stringent hybridization (25% instead of 50% formamide in the manufacturer's recommended hybridization buffer) was employed. Washing conditions also included 2×SSC containing 0.1% SDS at 500C instead of 1×SSC containing 0.1% SDS at 65° C.

The resultant human double-stranded 2045 bp cDNA homolog and encoded amino acid residue is shown in FIGS. 14-1 through 14-5 with the ATG start site beginning at nucleotide position 36 indicated with the boxed methionine (M) residue. The stop codon, TGA, is similarly boxed as shown on FIG. 14-4 at nucleotide positions 1773 to 1775 followed by the 3′ untranslated region. The top and bottom strands of the cDNA sequence are also respectively listed as SEQ ID Nos 42 and 44. The encoded amino acid sequence is shown in with the top strand in SEQ ID NO 42 and again by itself in SEQ ID NO 43.

E. Preparation of Expressed Recombinant the Fatty Acid Amide Hydrolase Cis-9,10-Octadecenoamidase:

For preparing recombinant FAAH proteins for use in this invention, the rat, mouse and human cDNAs obtained above were separately cloned into the eukaryotic expression vector pcDNA3 for transient expression studies in COS-7 cells.

For preparing the rat, mouse and human FAAH recombinant protein, the corresponding FAAH cDNAs were excised from the Bluescript II vectors and separately ligated into the eukaryotic expression vector, pcDNA3 (Invitrogen, San Diego, Calif.). 100 mm dishes of COS-7 cells were grown at 37° C. to 70% confluency in complete medium (DMEM with L-glutamine, non-essential amino acids, sodium pyruvate and fetal bovine serum). The COS-7 cells were'then washed with serum-free medium and treated with 5 ml of transfection solution (5-6 μg of FAAH-pcDNA3 vector were preincubated with transfectamine (Gibco-BRL) for 30 minutes in 1 ml of serum free medium, then diluted to a final volume of 5 ml with serum free medium). The COS-7 cells were incubated at 37° C. for 5 hours, at which point 10 ml of complete medium was added to the cells and incubation was continued at 37° C. for 12 hours. The transfection solution was then aspirated away from the COS-7 cells, and the cells were incubated in a fresh batch of complete medium for another 24 hours. The COS-7 cells were harvested with a cell scraper, pelleted at low speed, washed twice with 1 mM NaHCO₃, and resuspended in 200 μl of 1 mM NaHCO₃. The resuspended COS-7 cells were dounce homogenized 12 times and 20 μl of the resulting cell extract was used to assay for oleamide hydrolase activity (assay is detailed above in Section B6) with the results as described below in Section F. Control COS-7 cells were prepared identically except that the pcDNA3 vector used for transfection contained the FAAH cDNA in reverse orientation.

The resultant expressed recombinant FAAH proteins for rat, human and mouse are then used as described below to assess specificity and enzymatic activity.

F. Fatty Acid Amide Hydrolase Specifificty and Activity of the Expressed Recombinant Fatty Acid Amide Hydrolases

As described above, the transfected COS-7 cells were lysed to generate a cell extract for each of the recombinant expressed rat, mouse and human FAAH proteins of this invention.

While untransfected COS-7 cells contained negligible amounts of oleamide hydrolase activity, COS-7 cells transfected with the rat FAAH cDNA expressed high levels of oleamide hydrolase activity (FIG. 15A). The assay was performed as described in Section B where by TLC the conversion of oleamide to oleic acid was assessed. As shown in FIG. 15A, COS-7 cells transiently tranfected with rat oleamide hydrolase cDNA in expression vector pcDNA 3 shown in lane 3 but not in untransfected COS-7 cells (lane 1) or control transfected cells (lane 2, transfected with pcDNA3 containing the oleamide hydrolase cDNA in reverse orientation), were effective at converting labeled oleamide to oleic acid. Similar results were obtained with COS-7 cells transiently transfected with human oleamide hydrolase as shown in FIG. 16 where the conversion to oleic acid is seen only in lane 2 as compared to control COS-7 cells in lane 1.

This enzyme activity, like the rat liver plasma membrane oleamide hydrolase activity, was inhibited by trifluoromethyl ketone as evidenced in FIG. 15B as shown in lane 2 of the figure the rat oleamide hydrolase-transfected COS-7 cells in the presence of 50 μM trifluoromethyl ketone as compared to the untreated extract in lane 1.

To confirm specificity of the expressed recombinant proteins, Western blot analyses with anti-FAAH polyclonal antibodies alone or in the presence of competing peptides were performed. Samples of cell extract from rat FAAH-transfected and untransfected COS-7 cells with approximately equal protein amounts were heated to 65° C. for 10 minutes in loading buffer with 2% SDS and 5% β-mercaptoethanol. The samples indicated above were then run on an 8-16% polyacrylamide gradient Tris-glycine gel, and transferred to nitrocellulose for Western blotting. The nitrocellulose blot was blocked with 5% Blotto in TBS-Tween overnight at 4° C., and then incubated with polyclonal antibodies generated against peptide 2 as previously described (15 μg/ml in TBS-Tween) generated against an internal FAAH peptide sequence for 2 hours at 25° C. The blot was then washed in TBS-Tween (0.1%), incubated with a secondary antibody-horseradish peroxidase conjugate for 30 minutes at 25° C., washed again in TBS-Tween, and developed with Stable Peroxide Solution and Luminol/Enhancer Solution (Pierce). Peptide competition experiments were conducted by preincubating 1000-fold molar excess of the peptide antigen corresponding to peptide 2 as previously described with polyclonal antibodies for 30 minutes prior to addition of antibodies to the blot.

Western blotting of the rat cDNA transfected COS-7 cell extract with polyclonal antibodies generated against the internal peptide 2 sequence of FAAH showed a 60-65 kDa immunoreactive band that comigrated with affinity-isolated FAAH on SDS-PAGE (FIG. 15C). Untransfected COS-7 cell extract contained no detectable immunoreactive protein band of this size. Additionally, the immunoreactivity of the 60-65 kDa protein was effectively competed away by preincubation of the antibodies with excess peptide antigen (FIG. 15C), while the trace quantities of cross reactive protein observed in both the transfected and untransfected COS-7 cell extracts were not competed by this peptide.

Previous work suggested that the enzyme activity that hydrolyzes oleamide may be the same activity that converts anandamide (arachidonyl ethanolamide) to arachidonic acid. Therefore, COS-7 cells transfected with the rat FAAH cDNA were assayed for anandamide hydrolase activity. To assess the enzymatic activity of the expressed recombinant fatty acid amide hydrolases of this invention on labeled anandamide, the following enzymatic assay was performed. ¹⁴C-anandamide was synthesized as follows: 12.5 μCi (specific activity of 50 μCi/AM) of ¹⁴C arachidonic acid (Moravek Biochemicals) was dissolved in 100 μl CH₂Cl₂, cooled to 0° C., and treated with excess oxalyl chloride. The reaction mixture was stirred at 25° C. for 6 hours, after which time the solvent was evaporated. The remaining residue was cooled to 0° C., treated with a large excess of ethanolamine, and stirred at 25° C. for 15 minutes. The reaction mixture was then partitioned between ethyl acetate and 2 M HCl, and the organic layer was washed with water and then evaporated to dryness. The resulting ¹⁴C-anandamide was diluted with unlabeled anandamide to a final specific activity of 5 μCi/μM in ethanol. Approximately 1 μCi of ¹⁴C-anandamide and 20 μl of dounce homogenized COS-7 cell extract were used for each anandamide hydrolase assay as detailed above for the oleamide hydrolase assays. Briefly, FAAH hydrolysis assays were conducted in triplicate with 100 μM substrate, 35 μg of rat transfected COS-7 cell protein for 5 minutes at 37° C. (except in the case of stearic amide, where due to low solubility, 20 μM substrate comparison to oleamide were conducted). Products were separated on TLC as described previously, scraped into scintillation fluid, and radioactivity was quantitated by scintillation counting. Substrate hydrolysis in the presence of equal amounts of untransfected COS-7 cell protein extract served as background control in all cases and was substracted from FAAH hydrolysis rates to give the data as presented below.

The results of the anandamide assays showed that while untransfected COS-7 cells contained negligible quantities of anandamide hydrolase activity, transfected COS-7 cells produced high levels of anandamide hydrolase activity (FIG. 17). Thus, FAAH has the capacity to hydrolyze both oleamide and anandamide, indicating that the amidase may act as a general degradative enzyme for the fatty acid amide family of signaling molecules. The substrate promiscuity of FAAH is reminiscent of the monoamine oxidase enzymes which serve to oxidize a variety of amine-containing neurotransmitters.

To further assess the substrate specificity spectrum of enyzmatic hydolytic activity of the recombinant expressed proteins of this invention, other ¹⁴C-labeled fatty acid amides were synthesized as described in Section B6 and above for ¹⁴C-oleamide, with the exception of anandamide as described.

The results showed that while recombinant expressed rat FAAH catalyzes the hydrolysis of oleamide and anandamide at approximately equal rates, FAAH does discriminate among fatty acid amides, as FAAH hydroylzes other representative fatty acid amides, including myristic amide, palmitic amide and stearic amide at a significantly reduced rate as compared to that seen with oleamide or anandamide as shown in Table 1 below. Where indicated in the table the anandamide and oleamide hydrolysis rates are considered to be 100% of FAAH activity to which other fatty acid amide hydrolysis rates are compared.

TABLE 1 Substrate Rate of Hydrolysis* % Anandamide (100 μM) 333 +/− 30 100 Oleamide (100 μM) 242 +/− 20 72.6 Myristic Amide (100 μM)  81 +/− 7 24.3 Palmitic Amide (100 μM)  33 +/− 2 9.9 Oleamide (20 μM)  41 +/− 2 100 Stearic Amide (20 μM)  2.3 +/− 1 5.8 * Rate is measured in nmol/min/mg for each

Comparable assays are performed with the mouse and human recombinant homologs to the rat enzyme as used above.

Thus, as shown above, the rat FAAH enzyme was not without substrate preference, albeit it did exhibit activity against a number of amide substrates. The degree to which FAAH showed substrate selectivity is best exemplified by the nearly twenty fold rate difference between the enzyme's hydrolysis of oleamide and steric amide, two compounds that only differ by a single degree of unsaturation at the Δ9 position. This pattern was also confirmed with assays with the inhibitor trifluoromethyl ketone that was a twenty fold stronger inhibitor of FAAH than for the corresponding trifluoromethyl ketone analog of stearic amide. Thus, FAAH significantly favors the bent alkyl chain of oleamide over the straight alkyl chain of stearic amide.

A deletion mutant for generating a soluble form of the FAAH molecules of this invention was also prepared. A construct was created in which the putative transmembrane domain was deleted resulting in a truncated FAAH beginning at amino acid residue 30 of the encoded protein rather than 1. To prepare this construct, the following primers were designed for PCR amplification of the 5′ end of rat FAAH cDNA lacking the first 140 bp encoding the amino terminal 30 amino acids of FAAH. The 5′ and 3′ primers had the respective nucleotide sequences 5′GCGGTACCATGCGATGGACCGGGCGC3′ (SEQ ID NO 45) encoding amino acids 30-35 and containing a KpnI site and an artificial stop codon and 5′GGTCTGGCCAAAGAGAGG3′ (SEQ ID NO 46) where its reverse complement encodes amino acids 199-204.

The amplified transmembrane deleted rat FAAH cDNA fragment was then digested with the appropriate restriction enzymes (KpnI and HindIII) and cloned into the similarly digested FAAH-pBluescript vector replacing the original cDNA 5′ end. The deleted construct was confirmed by sequencing and then excised and transferred to pcDNA3 for expression studies as described herein.

For expression, the transfected COS-7 cell extract was separated into soluble and membrane fractions as follows: the extract was spun at 2500 rpm for 5 minutes at 25° C. and the supernatant was transferred to an airfuge tube and spun in an ultracentrifuge (30 psi for 40 minutes at 4° C.) for preparing soluble supernatant. The pellet contained the membrane bound fraction that was then resuspended in a volume of 1 mM NaHCO₃ equal to the volume of the supernatant.

The transmembrane-deleted expressed recombinant FAAH was functional in COS-7 cell expression assays as described above. The mouse and human transmembrane truncation homologs of the rat cDNA are similarly prepared and used in practicing this invention.

Given the increasing number of studies demonstrating biological activities for various members of the fatty acid amide family of signaling molecules, the discovery of a family of fatty acid amide hydrolases (FAAH) having homology between rat, mouse and human as described herein provides a valuable invention for ongoing studies dedicated to understanding the regulation, mechanism, and pharmacology of the metabolic process that inactivates the fatty acid amides. In addition, the cloned FAAH gene in conjunction with potent FAAH inhibitors provides the ability in both elucidating the physiological pathways affected by the fatty acid amide family and developing systematic approaches towards the pharmacological intervention of these biological processes.

54 783 base pairs nucleic acid double linear cDNA NO NO not provided CDS 1..783 1 AGC CCA GGA GGT TCC TCA GGG GGT GAG GGG GCT CTC ATT GGA TCT GGA 48 Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly Ser Gly 1 5 10 15 GGT TCC CCT CTG GGT TTA GGC ACT GAC ATT GGC GGC AGC ATC CGG TTC 96 Gly Ser Pro Leu Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Phe 20 25 30 CCT TCT GCC TTC TGC GGC ATC TGT GGC CTC AAG CCT ACT GGC AAC CGC 144 Pro Ser Ala Phe Cys Gly Ile Cys Gly Leu Lys Pro Thr Gly Asn Arg 35 40 45 CTC AGC AAG AGT GGC CTG AAG GGC TGT GTC TAT GGA CAG ACG GCA GTG 192 Leu Ser Lys Ser Gly Leu Lys Gly Cys Val Tyr Gly Gln Thr Ala Val 50 55 60 CAG CTT TCT CTT GGC CCC ATG GCC CGG GAT GTG GAG AGC CTG GCG CTA 240 Gln Leu Ser Leu Gly Pro Met Ala Arg Asp Val Glu Ser Leu Ala Leu 65 70 75 80 TGC CTG AAA GCT CTA CTG TGT GAG CAC TTG TTC ACC TTG GAC CCT ACC 288 Cys Leu Lys Ala Leu Leu Cys Glu His Leu Phe Thr Leu Asp Pro Thr 85 90 95 GTG CCT CCC TTT CCC TTC AGA GAG GAG GTC TAT AGA AGT TCT AGA CCC 336 Val Pro Pro Phe Pro Phe Arg Glu Glu Val Tyr Arg Ser Ser Arg Pro 100 105 110 CTG CGT GTG GGG TAC TAT GAG ACT GAC AAC TAT ACC ATG CCC AGC CCA 384 Leu Arg Val Gly Tyr Tyr Glu Thr Asp Asn Tyr Thr Met Pro Ser Pro 115 120 125 GCT ATG AGG AGG GCT CTG ATA GAG ACC AAG CAG AGA CTT GAG GCT GCT 432 Ala Met Arg Arg Ala Leu Ile Glu Thr Lys Gln Arg Leu Glu Ala Ala 130 135 140 GGC CAC ACG CTG ATT CCC TTC TTA CCC AAC AAC ATA CCC TAC GCC CTG 480 Gly His Thr Leu Ile Pro Phe Leu Pro Asn Asn Ile Pro Tyr Ala Leu 145 150 155 160 GAG GTC CTG TCT GCG GGC GGC CTG TTC AGT GAC GGT GGC CGC AGT TTT 528 Glu Val Leu Ser Ala Gly Gly Leu Phe Ser Asp Gly Gly Arg Ser Phe 165 170 175 CTC CAA AAC TTC AAA GGT GAC TTT GTG GAT CCC TGC TTG GGA GAC CTG 576 Leu Gln Asn Phe Lys Gly Asp Phe Val Asp Pro Cys Leu Gly Asp Leu 180 185 190 ATC TTA ATT CTG AGG CTG CCC AGC TGG TTT AAA AGA CTG CTG AGC CTC 624 Ile Leu Ile Leu Arg Leu Pro Ser Trp Phe Lys Arg Leu Leu Ser Leu 195 200 205 CTG CTG AAG CCT CTG TTT CCT CGG CTG GCA GCC TTT CTC AAC AGT ATG 672 Leu Leu Lys Pro Leu Phe Pro Arg Leu Ala Ala Phe Leu Asn Ser Met 210 215 220 CGT CCT CGG TCA GCT GAA AAG CTG TGG AAA CTG CAG CAT GAG ATT GAG 720 Arg Pro Arg Ser Ala Glu Lys Leu Trp Lys Leu Gln His Glu Ile Glu 225 230 235 240 ATG TAT CGC CAG TCT GTG ATT GCC CAG TGG AAA GCG ATG AAC TTG GAT 768 Met Tyr Arg Gln Ser Val Ile Ala Gln Trp Lys Ala Met Asn Leu Asp 245 250 255 GTG CTG CTG ACC TAA 783 Val Leu Leu Thr 260 260 amino acids amino acid linear protein not provided 2 Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly Ser Gly 1 5 10 15 Gly Ser Pro Leu Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Phe 20 25 30 Pro Ser Ala Phe Cys Gly Ile Cys Gly Leu Lys Pro Thr Gly Asn Arg 35 40 45 Leu Ser Lys Ser Gly Leu Lys Gly Cys Val Tyr Gly Gln Thr Ala Val 50 55 60 Gln Leu Ser Leu Gly Pro Met Ala Arg Asp Val Glu Ser Leu Ala Leu 65 70 75 80 Cys Leu Lys Ala Leu Leu Cys Glu His Leu Phe Thr Leu Asp Pro Thr 85 90 95 Val Pro Pro Phe Pro Phe Arg Glu Glu Val Tyr Arg Ser Ser Arg Pro 100 105 110 Leu Arg Val Gly Tyr Tyr Glu Thr Asp Asn Tyr Thr Met Pro Ser Pro 115 120 125 Ala Met Arg Arg Ala Leu Ile Glu Thr Lys Gln Arg Leu Glu Ala Ala 130 135 140 Gly His Thr Leu Ile Pro Phe Leu Pro Asn Asn Ile Pro Tyr Ala Leu 145 150 155 160 Glu Val Leu Ser Ala Gly Gly Leu Phe Ser Asp Gly Gly Arg Ser Phe 165 170 175 Leu Gln Asn Phe Lys Gly Asp Phe Val Asp Pro Cys Leu Gly Asp Leu 180 185 190 Ile Leu Ile Leu Arg Leu Pro Ser Trp Phe Lys Arg Leu Leu Ser Leu 195 200 205 Leu Leu Lys Pro Leu Phe Pro Arg Leu Ala Ala Phe Leu Asn Ser Met 210 215 220 Arg Pro Arg Ser Ala Glu Lys Leu Trp Lys Leu Gln His Glu Ile Glu 225 230 235 240 Met Tyr Arg Gln Ser Val Ile Ala Gln Trp Lys Ala Met Asn Leu Asp 245 250 255 Val Leu Leu Thr 260 22 base pairs nucleic acid single linear cDNA NO NO not provided 3 CGGAATTCGG NGGNGARGGN GC 22 5 amino acids amino acid linear peptide internal not provided 4 Gly Gly Glu Gly Ala 1 5 31 amino acids amino acid linear peptide internal not provided 5 Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly Ser Gly Gly Ser 1 5 10 15 Pro Leu Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Phe Pro 20 25 30 15 amino acids amino acid linear peptide internal not provided 6 Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly Ser 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 7 Ala Leu Ile Gly Ser Gly Gly Ser Pro Leu Gly Leu Gly Thr Asp 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 8 Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Phe Pro Ser Ala 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 9 Arg Phe Pro Ser Ala Phe Cys Gly Ile Cys Gly Leu Lys Pro Thr 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 10 Gly Leu Lys Pro Thr Gly Asn Arg Leu Ser Lys Ser Gly Leu Lys 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 11 Lys Ser Gly Leu Lys Gly Cys Val Tyr Gly Gln Thr Ala Val Gln 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 12 Gln Thr Ala Val Gln Leu Ser Leu Gly Pro Met Ala Arg Asp Val 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 13 Met Ala Arg Asp Val Glu Ser Leu Ala Leu Cys Leu Lys Ala Leu 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 14 Cys Leu Lys Ala Leu Leu Cys Glu His Leu Phe Thr Leu Asp Pro 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 15 Phe Thr Leu Asp Pro Thr Val Pro Pro Phe Pro Phe Arg Glu Glu 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 16 Pro Phe Arg Glu Glu Val Tyr Arg Ser Ser Arg Pro Leu Arg Val 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 17 Arg Pro Leu Arg Val Gly Tyr Tyr Glu Thr Asp Asn Tyr Thr Met 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 18 Asp Asn Tyr Thr Met Pro Ser Pro Ala Met Arg Arg Ala Leu Ile 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 19 Arg Arg Ala Leu Ile Glu Thr Lys Gln Arg Leu Glu Ala Ala Gly 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 20 Leu Glu Ala Ala Gly His Thr Leu Ile Pro Phe Leu Pro Asn Asn 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 21 Phe Leu Pro Asn Asn Ile Pro Tyr Ala Leu Glu Val Leu Ser Ala 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 22 Glu Val Leu Ser Ala Gly Gly Leu Phe Ser Asp Gly Gly Arg Ser 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 23 Asp Gly Gly Arg Ser Phe Leu Gln Asn Phe Lys Gly Asp Phe Val 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 24 Lys Gly Asp Phe Val Asp Pro Cys Leu Gly Asp Leu Ile Leu Ile 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 25 Asp Leu Ile Leu Ile Leu Arg Leu Pro Ser Trp Phe Lys Arg Leu 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 26 Trp Phe Lys Arg Leu Leu Ser Leu Leu Leu Lys Pro Leu Phe Pro 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 27 Lys Pro Leu Phe Pro Arg Leu Ala Ala Phe Leu Asn Ser Met Arg 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 28 Leu Asn Ser Met Arg Pro Arg Ser Ala Glu Lys Leu Trp Lys Leu 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 29 Lys Leu Trp Lys Leu Gln His Glu Ile Glu Met Tyr Arg Gln Ser 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 30 Met Tyr Arg Gln Ser Val Ile Ala Gln Trp Lys Ala Met Asn Leu 1 5 10 15 15 amino acids amino acid linear peptide internal not provided 31 Lys Ala Met Asn Leu Asp Val Leu Leu Thr Pro Met Leu Gly Pro 1 5 10 15 14 amino acids amino acid linear peptide internal not provided 32 Pro Met Leu Gly Pro Ala Leu Asp Leu Asn Thr Pro Gly Arg 1 5 10 25 base pairs nucleic acid single linear cDNA NO NO not provided 33 CGGGATCCGG CATNGTRTAR TTRTC 25 6 amino acids amino acid linear peptide internal not provided 34 Asp Asn Tyr Thr Met Pro 1 5 2472 base pairs nucleic acid double linear cDNA NO NO not provided CDS 50..1789 35 GGTTTGTGCG AGCCGAGTTC TCTCGGGTGG CGGTCGGCTG CAGGAGATC ATG GTG 55 Met Val 1 CTG AGC GAA GTG TGG ACC ACG CTG TCT GGG GTC TCC GGG GTT TGC CTA 103 Leu Ser Glu Val Trp Thr Thr Leu Ser Gly Val Ser Gly Val Cys Leu 5 10 15 GCC TGC AGC TTG TTG TCG GCG GCG GTG GTC CTG CGA TGG ACC GGG CGC 151 Ala Cys Ser Leu Leu Ser Ala Ala Val Val Leu Arg Trp Thr Gly Arg 20 25 30 CAG AAG GCC CGG GGC GCG GCG ACC AGG GCG CGG CAG AAG CAG CGA GCC 199 Gln Lys Ala Arg Gly Ala Ala Thr Arg Ala Arg Gln Lys Gln Arg Ala 35 40 45 50 AGC CTG GAG ACC ATG GAC AAG GCG GTG CAG CGC TTC CGG CTG CAG AAT 247 Ser Leu Glu Thr Met Asp Lys Ala Val Gln Arg Phe Arg Leu Gln Asn 55 60 65 CCT GAC CTG GAC TCG GAG GCC TTG CTG ACC CTG CCC CTA CTC CAA CTG 295 Pro Asp Leu Asp Ser Glu Ala Leu Leu Thr Leu Pro Leu Leu Gln Leu 70 75 80 GTA CAG AAG TTA CAG AGT GGA GAG CTG TCC CCA GAG GCT GTG TTC TTT 343 Val Gln Lys Leu Gln Ser Gly Glu Leu Ser Pro Glu Ala Val Phe Phe 85 90 95 ACT TAC CTG GGA AAG GCC TGG GAA GTG AAC AAA GGG ACC AAC TGC GTG 391 Thr Tyr Leu Gly Lys Ala Trp Glu Val Asn Lys Gly Thr Asn Cys Val 100 105 110 ACC TCC TAT CTG ACC GAC TGT GAG ACT CAG CTG TCC CAG GCC CCA CGG 439 Thr Ser Tyr Leu Thr Asp Cys Glu Thr Gln Leu Ser Gln Ala Pro Arg 115 120 125 130 CAG GGC CTG CTC TAT GGT GTC CCT GTG AGC CTC AAG GAA TGC TTC AGC 487 Gln Gly Leu Leu Tyr Gly Val Pro Val Ser Leu Lys Glu Cys Phe Ser 135 140 145 TAC AAG GGC CAC GAC TCC ACA CTG GGC TTG AGC CTG AAT GAG GGC ATG 535 Tyr Lys Gly His Asp Ser Thr Leu Gly Leu Ser Leu Asn Glu Gly Met 150 155 160 CCA TCG GAA TCT GAC TGT GTG GTG GTG CAA GTG TTG AAG CTG CAG GGA 583 Pro Ser Glu Ser Asp Cys Val Val Val Gln Val Leu Lys Leu Gln Gly 165 170 175 GCT GTG CCC TTT GTG CAT ACC AAT GTC CCC CAG TCC ATG TTA AGC TTT 631 Ala Val Pro Phe Val His Thr Asn Val Pro Gln Ser Met Leu Ser Phe 180 185 190 GAC TGC AGT AAC CCT CTC TTT GGC CAG ACC ATG AAC CCA TGG AAG TCC 679 Asp Cys Ser Asn Pro Leu Phe Gly Gln Thr Met Asn Pro Trp Lys Ser 195 200 205 210 TCC AAG AGC CCA GGA GGT TCC TCA GGG GGT GAG GGG GCT CTC ATT GGA 727 Ser Lys Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly 215 220 225 TCT GGA GGT TCC CCT CTG GGT TTA GGC ACT GAC ATT GGC GGC AGC ATC 775 Ser Gly Gly Ser Pro Leu Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile 230 235 240 CGG TTC CCT TCT GCC TTC TGC GGC ATC TGT GGC CTC AAG CCT ACT GGC 823 Arg Phe Pro Ser Ala Phe Cys Gly Ile Cys Gly Leu Lys Pro Thr Gly 245 250 255 AAC CGC CTC AGC AAG AGT GGC CTG AAG GGC TGT GTC TAT GGA CAG ACG 871 Asn Arg Leu Ser Lys Ser Gly Leu Lys Gly Cys Val Tyr Gly Gln Thr 260 265 270 GCA GTG CAG CTT TCT CTT GGC CCC ATG GCC CGG GAT GTG GAG AGC CTG 919 Ala Val Gln Leu Ser Leu Gly Pro Met Ala Arg Asp Val Glu Ser Leu 275 280 285 290 GCG CTA TGC CTG AAA GCT CTA CTG TGT GAG CAC TTG TTC ACC TTG GAC 967 Ala Leu Cys Leu Lys Ala Leu Leu Cys Glu His Leu Phe Thr Leu Asp 295 300 305 CCT ACC GTG CCT CCC TTG CCC TTC AGA GAG GAG GTC TAT AGA AGT TCT 1015 Pro Thr Val Pro Pro Leu Pro Phe Arg Glu Glu Val Tyr Arg Ser Ser 310 315 320 AGA CCC CTG CGT GTG GGG TAC TAT GAG ACT GAC AAC TAT ACC ATG CCC 1063 Arg Pro Leu Arg Val Gly Tyr Tyr Glu Thr Asp Asn Tyr Thr Met Pro 325 330 335 AGC CCA GCT ATG AGG AGG GCT CTG ATA GAG ACC AAG CAG AGA CTT GAG 1111 Ser Pro Ala Met Arg Arg Ala Leu Ile Glu Thr Lys Gln Arg Leu Glu 340 345 350 GCT GCT GGC CAC ACG CTG ATT CCC TTC TTA CCC AAC AAC ATA CCC TAC 1159 Ala Ala Gly His Thr Leu Ile Pro Phe Leu Pro Asn Asn Ile Pro Tyr 355 360 365 370 GCC CTG GAG GTC CTG TCT GCG GGC GGC CTG TTC AGT GAC GGT GGC CGC 1207 Ala Leu Glu Val Leu Ser Ala Gly Gly Leu Phe Ser Asp Gly Gly Arg 375 380 385 AGT TTT CTC CAA AAC TTC AAA GGT GAC TTT GTG GAT CCC TGC TTG GGA 1255 Ser Phe Leu Gln Asn Phe Lys Gly Asp Phe Val Asp Pro Cys Leu Gly 390 395 400 GAC CTG ATC TTA ATT CTG AGG CTG CCC AGC TGG TTT AAA AGA CTG CTG 1303 Asp Leu Ile Leu Ile Leu Arg Leu Pro Ser Trp Phe Lys Arg Leu Leu 405 410 415 AGC CTC CTG CTG AAG CCT CTG TTT CCT CGG CTG GCA GCC TTT CTC AAC 1351 Ser Leu Leu Leu Lys Pro Leu Phe Pro Arg Leu Ala Ala Phe Leu Asn 420 425 430 AGT ATG CGT CCT CGG TCA GCT GAA AAG CTG TGG AAA CTG CAG CAT GAG 1399 Ser Met Arg Pro Arg Ser Ala Glu Lys Leu Trp Lys Leu Gln His Glu 435 440 445 450 ATT GAG ATG TAT CGC CAG TCT GTG ATT GCC CAG TGG AAA GCG ATG AAC 1447 Ile Glu Met Tyr Arg Gln Ser Val Ile Ala Gln Trp Lys Ala Met Asn 455 460 465 TTG GAT GTG CTG CTG ACC CCC ATG TTG GGC CCT GCT CTG GAT TTG AAC 1495 Leu Asp Val Leu Leu Thr Pro Met Leu Gly Pro Ala Leu Asp Leu Asn 470 475 480 ACA CCG GGC AGA GCC ACA GGG GCT ATC AGC TAC ACC GTT CTC TAC AAC 1543 Thr Pro Gly Arg Ala Thr Gly Ala Ile Ser Tyr Thr Val Leu Tyr Asn 485 490 495 TGC CTG GAC TTC CCT GCG GGG GTG GTG CCT GTC ACC ACT GTG ACC GCC 1591 Cys Leu Asp Phe Pro Ala Gly Val Val Pro Val Thr Thr Val Thr Ala 500 505 510 GAG GAC GAT GCC CAG ATG GAA CTC TAC AAA GGC TAC TTT GGG GAT ATC 1639 Glu Asp Asp Ala Gln Met Glu Leu Tyr Lys Gly Tyr Phe Gly Asp Ile 515 520 525 530 TGG GAC ATC ATC CTG AAG AAG GCC ATG AAA AAT AGT GTC GGT CTG CCT 1687 Trp Asp Ile Ile Leu Lys Lys Ala Met Lys Asn Ser Val Gly Leu Pro 535 540 545 GTG GCT GTG CAG TGC GTG GCT CTG CCC TGG CAG GAA GAG CTG TGT CTG 1735 Val Ala Val Gln Cys Val Ala Leu Pro Trp Gln Glu Glu Leu Cys Leu 550 555 560 AGG TTC ATG CGG GAG GTG GAA CAG CTG ATG ACC CCT CAA AAG CAG CCA 1783 Arg Phe Met Arg Glu Val Glu Gln Leu Met Thr Pro Gln Lys Gln Pro 565 570 575 TCG TGAGGGTCGT TCATCCGCCA GCTCTGGAGG ACCTAAGGCC CATGCGCTGT 1836 Ser 580 GCACTGTAGC CCCATGTATT CAGGAGCCAC CACCCACGAG GGAACGCCCA GCACAGGGAA 1896 GAGGTGTCTA CCTGCCCTCC CCTGGACTCC TGCAGCCACA ACCAAGTCTG GACCTTCCTC 1956 CCCGTTATGG TCTACTTTCC ATCCTGATTC CCTGCTTTTT ATGGCAGCCA GCAGGAATGA 2016 CGTGGGCCAA GGATCACCAA CATTCAAAAA CAATGCGTTT ATCTATTTTC TGGGTATCTC 2076 CATTAGGGCC CTGGGAACCA GAGTGCTGGG AAGGCTGTCC AGACCCTCCA GAGCTGGCTG 2136 TAACCACATC ACTCTCCTGC TCCAAAGCCT CCCTAGTTCT GTCACCCACA AGATAGACAC 2196 AGGGACATGT CCTTGGCACT TGACTCCTGT CCTTCCTTTC TTATTCAGAT TGACCCCAGC 2256 CTTGATGGAC CCTGCCCCTG CACTTCCTTC CTCAGTCCAC CTCTCTGCCG ACACGCCCTT 2316 TTTATGGCTC CTCTATTTGT TGTGGAGACA AGGTTTCTCT CAGTAGCCCT GGCTGTCCAG 2376 GACCTCACTC TGTAGATGAG GCTGGCTTTC AACTCACAAG GCTGCCTGCC TGGGTGCTGG 2436 GATTAAAGGC GTATGCCACC ACAAAGAAAA AAAAAA 2472 579 amino acids amino acid linear protein not provided 36 Met Val Leu Ser Glu Val Trp Thr Thr Leu Ser Gly Val Ser Gly Val 1 5 10 15 Cys Leu Ala Cys Ser Leu Leu Ser Ala Ala Val Val Leu Arg Trp Thr 20 25 30 Gly Arg Gln Lys Ala Arg Gly Ala Ala Thr Arg Ala Arg Gln Lys Gln 35 40 45 Arg Ala Ser Leu Glu Thr Met Asp Lys Ala Val Gln Arg Phe Arg Leu 50 55 60 Gln Asn Pro Asp Leu Asp Ser Glu Ala Leu Leu Thr Leu Pro Leu Leu 65 70 75 80 Gln Leu Val Gln Lys Leu Gln Ser Gly Glu Leu Ser Pro Glu Ala Val 85 90 95 Phe Phe Thr Tyr Leu Gly Lys Ala Trp Glu Val Asn Lys Gly Thr Asn 100 105 110 Cys Val Thr Ser Tyr Leu Thr Asp Cys Glu Thr Gln Leu Ser Gln Ala 115 120 125 Pro Arg Gln Gly Leu Leu Tyr Gly Val Pro Val Ser Leu Lys Glu Cys 130 135 140 Phe Ser Tyr Lys Gly His Asp Ser Thr Leu Gly Leu Ser Leu Asn Glu 145 150 155 160 Gly Met Pro Ser Glu Ser Asp Cys Val Val Val Gln Val Leu Lys Leu 165 170 175 Gln Gly Ala Val Pro Phe Val His Thr Asn Val Pro Gln Ser Met Leu 180 185 190 Ser Phe Asp Cys Ser Asn Pro Leu Phe Gly Gln Thr Met Asn Pro Trp 195 200 205 Lys Ser Ser Lys Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu 210 215 220 Ile Gly Ser Gly Gly Ser Pro Leu Gly Leu Gly Thr Asp Ile Gly Gly 225 230 235 240 Ser Ile Arg Phe Pro Ser Ala Phe Cys Gly Ile Cys Gly Leu Lys Pro 245 250 255 Thr Gly Asn Arg Leu Ser Lys Ser Gly Leu Lys Gly Cys Val Tyr Gly 260 265 270 Gln Thr Ala Val Gln Leu Ser Leu Gly Pro Met Ala Arg Asp Val Glu 275 280 285 Ser Leu Ala Leu Cys Leu Lys Ala Leu Leu Cys Glu His Leu Phe Thr 290 295 300 Leu Asp Pro Thr Val Pro Pro Leu Pro Phe Arg Glu Glu Val Tyr Arg 305 310 315 320 Ser Ser Arg Pro Leu Arg Val Gly Tyr Tyr Glu Thr Asp Asn Tyr Thr 325 330 335 Met Pro Ser Pro Ala Met Arg Arg Ala Leu Ile Glu Thr Lys Gln Arg 340 345 350 Leu Glu Ala Ala Gly His Thr Leu Ile Pro Phe Leu Pro Asn Asn Ile 355 360 365 Pro Tyr Ala Leu Glu Val Leu Ser Ala Gly Gly Leu Phe Ser Asp Gly 370 375 380 Gly Arg Ser Phe Leu Gln Asn Phe Lys Gly Asp Phe Val Asp Pro Cys 385 390 395 400 Leu Gly Asp Leu Ile Leu Ile Leu Arg Leu Pro Ser Trp Phe Lys Arg 405 410 415 Leu Leu Ser Leu Leu Leu Lys Pro Leu Phe Pro Arg Leu Ala Ala Phe 420 425 430 Leu Asn Ser Met Arg Pro Arg Ser Ala Glu Lys Leu Trp Lys Leu Gln 435 440 445 His Glu Ile Glu Met Tyr Arg Gln Ser Val Ile Ala Gln Trp Lys Ala 450 455 460 Met Asn Leu Asp Val Leu Leu Thr Pro Met Leu Gly Pro Ala Leu Asp 465 470 475 480 Leu Asn Thr Pro Gly Arg Ala Thr Gly Ala Ile Ser Tyr Thr Val Leu 485 490 495 Tyr Asn Cys Leu Asp Phe Pro Ala Gly Val Val Pro Val Thr Thr Val 500 505 510 Thr Ala Glu Asp Asp Ala Gln Met Glu Leu Tyr Lys Gly Tyr Phe Gly 515 520 525 Asp Ile Trp Asp Ile Ile Leu Lys Lys Ala Met Lys Asn Ser Val Gly 530 535 540 Leu Pro Val Ala Val Gln Cys Val Ala Leu Pro Trp Gln Glu Glu Leu 545 550 555 560 Cys Leu Arg Phe Met Arg Glu Val Glu Gln Leu Met Thr Pro Gln Lys 565 570 575 Gln Pro Ser 2472 base pairs nucleic acid double linear cDNA NO NO not provided 37 TTTTTTTTTT CTTTGTGGTG GCATACGCCT TTAATCCCAG CACCCAGGCA GGCAGCCTTG 60 TGAGTTGAAA GCCAGCCTCA TCTACAGAGT GAGGTCCTGG ACAGCCAGGG CTACTGAGAG 120 AAACCTTGTC TCCACAACAA ATAGAGGAGC CATAAAAAGG GCGTGTCGGC AGAGAGGTGG 180 ACTGAGGAAG GAAGTGCAGG GGCAGGGTCC ATCAAGGCTG GGGTCAATCT GAATAAGAAA 240 GGAAGGACAG GAGTCAAGTG CCAAGGACAT GTCCCTGTGT CTATCTTGTG GGTGACAGAA 300 CTAGGGAGGC TTTGGAGCAG GAGAGTGATG TGGTTACAGC CAGCTCTGGA GGGTCTGGAC 360 AGCCTTCCCA GCACTCTGGT TCCCAGGGCC CTAATGGAGA TACCCAGAAA ATAGATAAAC 420 GCATTGTTTT TGAATGTTGG TGATCCTTGG CCCACGTCAT TCCTGCTGGC TGCCATAAAA 480 AGCAGGGAAT CAGGATGGAA AGTAGACCAT AACGGGGAGG AAGGTCCAGA CTTGGTTGTG 540 GCTGCAGGAG TCCAGGGGAG GGCAGGTAGA CACCTCTTCC CTGTGCTGGG CGTTCCCTCG 600 TGGGTGGTGG CTCCTGAATA CATGGGGCTA CAGTGCACAG CGCATGGGCC TTAGGTCCTC 660 CAGAGCTGGC GGATGAACGA CCCTCACGAT GGCTGCTTTT GAGGGGTCAT CAGCTGTTCC 720 ACCTCCCGCA TGAACCTCAG ACACAGCTCT TCCTGCCAGG GCAGAGCCAC GCACTGCACA 780 GCCACAGGCA GACCGACACT ATTTTTCATG GCCTTCTTCA GGATGATGTC CCAGATATCC 840 CCAAAGTAGC CTTTGTAGAG TTCCATCTGG GCATCGTCCT CGGCGGTCAC AGTGGTGACA 900 GGCACCACCC CCGCAGGGAA GTCCAGGCAG TTGTAGAGAA CGGTGTAGCT GATAGCCCCT 960 GTGGCTCTGC CCGGTGTGTT CAAATCCAGA GCAGGGCCCA ACATGGGGGT CAGCAGCACA 1020 TCCAAGTTCA TCGCTTTCCA CTGGGCAATC ACAGACTGGC GATACATCTC AATCTCATGC 1080 TGCAGTTTCC ACAGCTTTTC AGCTGACCGA GGACGCATAC TGTTGAGAAA GGCTGCCAGC 1140 CGAGGAAACA GAGGCTTCAG CAGGAGGCTC AGCAGTCTTT TAAACCAGCT GGGCAGCCTC 1200 AGAATTAAGA TCAGGTCTCC CAAGCAGGGA TCCACAAAGT CACCTTTGAA GTTTTGGAGA 1260 AAACTGCGGC CACCGTCACT GAACAGGCCG CCCGCAGACA GGACCTCCAG GGCGTAGGGT 1320 ATGTTGTTGG GTAAGAAGGG AATCAGCGTG TGGCCAGCAG CCTCAAGTCT CTGCTTGGTC 1380 TCTATCAGAG CCCTCCTCAT AGCTGGGCTG GGCATGGTAT AGTTGTCAGT CTCATAGTAC 1440 CCCACACGCA GGGGTCTAGA ACTTCTATAG ACCTCCTCTC TGAAGGGCAA GGGAGGCACG 1500 GTAGGGTCCA AGGTGAACAA GTGCTCACAC AGTAGAGCTT TCAGGCATAG CGCCAGGCTC 1560 TCCACATCCC GGGCCATGGG GCCAAGAGAA AGCTGCACTG CCGTCTGTCC ATAGACACAG 1620 CCCTTCAGGC CACTCTTGCT GAGGCGGTTG CCAGTAGGCT TGAGGCCACA GATGCCGCAG 1680 AAGGCAGAAG GGAACCGGAT GCTGCCGCCA ATGTCAGTGC CTAAACCCAG AGGGGAACCT 1740 CCAGATCCAA TGAGAGCCCC CTCACCCCCT GAGGAACCTC CTGGGCTCTT GGAGGACTTC 1800 CATGGGTTCA TGGTCTGGCC AAAGAGAGGG TTACTGCAGT CAAAGCTTAA CATGGACTGG 1860 GGGACATTGG TATGCACAAA GGGCACAGCT CCCTGCAGCT TCAACACTTG CACCACCACA 1920 CAGTCAGATT CCGATGGCAT GCCCTCATTC AGGCTCAAGC CCAGTGTGGA GTCGTGGCCC 1980 TTGTAGCTGA AGCATTCCTT GAGGCTCACA GGGACACCAT AGAGCAGGCC CTGCCGTGGG 2040 GCCTGGGACA GCTGAGTCTC ACAGTCGGTC AGATAGGAGG TCACGCAGTT GGTCCCTTTG 2100 TTCACTTCCC AGGCCTTTCC CAGGTAAGTA AAGAACACAG CCTCTGGGGA CAGCTCTCCA 2160 CTCTGTAACT TCTGTACCAG TTGGAGTAGG GGCAGGGTCA GCAAGGCCTC CGAGTCCAGG 2220 TCAGGATTCT GCAGCCGGAA GCGCTGCACC GCCTTGTCCA TGGTCTCCAG GCTGGCTCGC 2280 TGCTTCTGCC GCGCCCTGGT CGCCGCGCCC CGGGCCTTCT GGCGCCCGGT CCATCGCAGG 2340 ACCACCGCCG CCGACAACAA GCTGCAGGCT AGGCAAACCC CGGAGACCCC AGACAGCGTG 2400 GTCCACACTT CGCTCAGCAC CATGATCTCC TGCAGCCGAC CGCCACCCGA GAGAACTCGG 2460 CTCGCACAAA CC 2472 6 amino acids amino acid linear peptide internal not provided 38 Pro Pro Leu Pro Xaa Arg 1 5 1959 base pairs nucleic acid double linear cDNA NO NO not provided CDS 1..1746 39 TGG GTC ATG GTG CTG AGC GAA GTG TGG ACC GCG CTG TCT GGA CTC TCC 48 Trp Val Met Val Leu Ser Glu Val Trp Thr Ala Leu Ser Gly Leu Ser 1 5 10 15 GGG GTT TGC CTA GCC TGC AGC TTG CTG TCG GCG GCG GTG GTC CTG CGA 96 Gly Val Cys Leu Ala Cys Ser Leu Leu Ser Ala Ala Val Val Leu Arg 20 25 30 TGG ACC AGG AGC CAG ACC GCC CGG GGC GCG GTG ACC AGG GCG CGG CAG 144 Trp Thr Arg Ser Gln Thr Ala Arg Gly Ala Val Thr Arg Ala Arg Gln 35 40 45 AAG CAG CGA GCC GGC CTG GAG ACC ATG GAC AAG GCG GTG CAG CGC TTC 192 Lys Gln Arg Ala Gly Leu Glu Thr Met Asp Lys Ala Val Gln Arg Phe 50 55 60 CGG CTG CAG AAT CCT GAC CTG GAT TCA GAG GCC TTG CTG GCT CTG CCC 240 Arg Leu Gln Asn Pro Asp Leu Asp Ser Glu Ala Leu Leu Ala Leu Pro 65 70 75 80 CTG CTC CAA CTG GTA CAG AAG TTA CAG AGT GGG GAA CTG TCC CCA GAA 288 Leu Leu Gln Leu Val Gln Lys Leu Gln Ser Gly Glu Leu Ser Pro Glu 85 90 95 GCT GTG CTC TTT ACC TAC CTG GGA AAG GCC TGG GAA GTG AAC AAA GGG 336 Ala Val Leu Phe Thr Tyr Leu Gly Lys Ala Trp Glu Val Asn Lys Gly 100 105 110 ACC AAC TGT GTG ACC TCC TAT CTG ACT GAC TGT GAG ACT CAG CTG TCC 384 Thr Asn Cys Val Thr Ser Tyr Leu Thr Asp Cys Glu Thr Gln Leu Ser 115 120 125 CAG GCC CCA CGG CAG GGC CTG CTC TAT GGC GTC CCC GTG AGC CTC AAG 432 Gln Ala Pro Arg Gln Gly Leu Leu Tyr Gly Val Pro Val Ser Leu Lys 130 135 140 GAA TGC TTC AGC TAC AAG GGC CAT GCT TCC ACA CTG GGC TTA AGT TTG 480 Glu Cys Phe Ser Tyr Lys Gly His Ala Ser Thr Leu Gly Leu Ser Leu 145 150 155 160 AAC GAG GGT GTG ACA TCG GAG AGT GAC TGT GTG GTG GTG CAG GTA CTG 528 Asn Glu Gly Val Thr Ser Glu Ser Asp Cys Val Val Val Gln Val Leu 165 170 175 AAG CTG CAG GGA GCT GTG CCC TTT GTG CAC ACC AAC GTC CCC CAG TCC 576 Lys Leu Gln Gly Ala Val Pro Phe Val His Thr Asn Val Pro Gln Ser 180 185 190 ATG CTA AGC TAT GAC TGC AGT AAC CCC CTC TTT GGC CAG ACC ATG AAC 624 Met Leu Ser Tyr Asp Cys Ser Asn Pro Leu Phe Gly Gln Thr Met Asn 195 200 205 CCG TGG AAG CCC TCC AAG AGT CCA GGA GGT TCC TCA GGG GGT GAG GGG 672 Pro Trp Lys Pro Ser Lys Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly 210 215 220 GCT CTC ATT GGA TCT GGA GGC TCC CCT CTG GGT TTA GGC ACT GAC ATC 720 Ala Leu Ile Gly Ser Gly Gly Ser Pro Leu Gly Leu Gly Thr Asp Ile 225 230 235 240 GGC GGC AGC ATC CGG TTC CCT TCT GCC TTC TGT GGC ATC TGT GGC CTC 768 Gly Gly Ser Ile Arg Phe Pro Ser Ala Phe Cys Gly Ile Cys Gly Leu 245 250 255 AAG CCT ACT GGG AAC CGC CTC AGC AAG AGT GGC CTG AAG AGC TGT GTT 816 Lys Pro Thr Gly Asn Arg Leu Ser Lys Ser Gly Leu Lys Ser Cys Val 260 265 270 TAT GGA CAG ACA GCA GTG CAG CTT TCT GTT GGC CCC ATG GCA CGG GAT 864 Tyr Gly Gln Thr Ala Val Gln Leu Ser Val Gly Pro Met Ala Arg Asp 275 280 285 GTG GAT AGC CTG GCA TTG TGC ATG AAA GCC CTA CTT TGT GAG GAT TTG 912 Val Asp Ser Leu Ala Leu Cys Met Lys Ala Leu Leu Cys Glu Asp Leu 290 295 300 TTC CGC TTG GAC TCC ACC ATC CCC CCC TTG CCC TTC AGG GAG GAG ATC 960 Phe Arg Leu Asp Ser Thr Ile Pro Pro Leu Pro Phe Arg Glu Glu Ile 305 310 315 320 TAC AGA AGT TCT CGA CCC CTT CGT GTG GGA TAC TAT GAA ACT GAC AAC 1008 Tyr Arg Ser Ser Arg Pro Leu Arg Val Gly Tyr Tyr Glu Thr Asp Asn 325 330 335 TAC ACC ATG CCC ACT CCA GCC ATG AGG AGG GCT GTG ATG GAG ACC AAG 1056 Tyr Thr Met Pro Thr Pro Ala Met Arg Arg Ala Val Met Glu Thr Lys 340 345 350 CAG AGT CTC GAG GCT GCT GGC CAC ACG CTG GTC CCC TTC TTA CCA AAC 1104 Gln Ser Leu Glu Ala Ala Gly His Thr Leu Val Pro Phe Leu Pro Asn 355 360 365 AAC ATA CCT TAT GCC CTG GAG GTC CTG TCG GCA GGT GGG CTG TTC AGT 1152 Asn Ile Pro Tyr Ala Leu Glu Val Leu Ser Ala Gly Gly Leu Phe Ser 370 375 380 GAT GGT GGC TGC TCT TTT CTC CAA AAC TTC AAA GGC GAC TTT GTG GAT 1200 Asp Gly Gly Cys Ser Phe Leu Gln Asn Phe Lys Gly Asp Phe Val Asp 385 390 395 400 CCC TGC TTG GGG GAC CTG GTC TTA GTG CTG AAG CTG CCC AGG TGG TTT 1248 Pro Cys Leu Gly Asp Leu Val Leu Val Leu Lys Leu Pro Arg Trp Phe 405 410 415 AAA AAA CTG CTG AGC TTC CTG CTG AAG CCT CTG TTT CCT CGG CTG GCA 1296 Lys Lys Leu Leu Ser Phe Leu Leu Lys Pro Leu Phe Pro Arg Leu Ala 420 425 430 GCC TTT CTC AAC AGT ATG TGT CCT CGG TCA GCC GAA AAG CTG TGG GAA 1344 Ala Phe Leu Asn Ser Met Cys Pro Arg Ser Ala Glu Lys Leu Trp Glu 435 440 445 CTG CAG CAT GAG ATT GAG ATG TAT CGC CAG TCC GTC ATT GCC CAG TGG 1392 Leu Gln His Glu Ile Glu Met Tyr Arg Gln Ser Val Ile Ala Gln Trp 450 455 460 AAG GCA ATG AAC TTG GAC GTG GTG CTA ACC CCC ATG CTG GGT CCT GCT 1440 Lys Ala Met Asn Leu Asp Val Val Leu Thr Pro Met Leu Gly Pro Ala 465 470 475 480 CTG GAT TTG AAC ACA CCG GGC AGA GCC ACA GGG GCT ATC AGC TAC ACT 1488 Leu Asp Leu Asn Thr Pro Gly Arg Ala Thr Gly Ala Ile Ser Tyr Thr 485 490 495 GTT CTC TAT AAC TGC CTG GAC TTC CCT GCG GGG GTG GTG CCT GTC ACC 1536 Val Leu Tyr Asn Cys Leu Asp Phe Pro Ala Gly Val Val Pro Val Thr 500 505 510 ACT GTG ACC GCT GAG GAC GAT GCC CAG ATG GAA CAC TAC AAA GGC TAC 1584 Thr Val Thr Ala Glu Asp Asp Ala Gln Met Glu His Tyr Lys Gly Tyr 515 520 525 TTT GGG GAT ATG TGG GAC AAC ATT CTG AAG AAG GGC ATG AAA AAG GGT 1632 Phe Gly Asp Met Trp Asp Asn Ile Leu Lys Lys Gly Met Lys Lys Gly 530 535 540 ATA GGC CTG CCT GTG GCT GTG CAG TGC GTG GCT CTG CCC TGG CAG GAA 1680 Ile Gly Leu Pro Val Ala Val Gln Cys Val Ala Leu Pro Trp Gln Glu 545 550 555 560 GAG CTG TGT CTG CGG TTC ATG CGG GAG GTG GAA CGG CTG ATG ACC CCT 1728 Glu Leu Cys Leu Arg Phe Met Arg Glu Val Glu Arg Leu Met Thr Pro 565 570 575 GAA AAG CGG CCA TCT TGAGGGTCAT TCATCTGCCC AGCTCTGGAG GACCTAAGGC 1783 Glu Lys Arg Pro Ser 580 CCATGCGCTC TGCACTGCAG CCCCATCTAT TCAGGATCCT GCCACCCATG AGGAGATGCC 1843 CAGCACGGGA AGAGGCAACC ACCTGCCCTC CCCTGGACTC CTACAGAAAC CCAGGACATG 1903 CCCTCCATAA CCAAGTCTGG ACCAGCTCCC CCGGAATTCC TGCAGCCCGG GGGATC 1959 581 amino acids amino acid linear protein not provided 40 Trp Val Met Val Leu Ser Glu Val Trp Thr Ala Leu Ser Gly Leu Ser 1 5 10 15 Gly Val Cys Leu Ala Cys Ser Leu Leu Ser Ala Ala Val Val Leu Arg 20 25 30 Trp Thr Arg Ser Gln Thr Ala Arg Gly Ala Val Thr Arg Ala Arg Gln 35 40 45 Lys Gln Arg Ala Gly Leu Glu Thr Met Asp Lys Ala Val Gln Arg Phe 50 55 60 Arg Leu Gln Asn Pro Asp Leu Asp Ser Glu Ala Leu Leu Ala Leu Pro 65 70 75 80 Leu Leu Gln Leu Val Gln Lys Leu Gln Ser Gly Glu Leu Ser Pro Glu 85 90 95 Ala Val Leu Phe Thr Tyr Leu Gly Lys Ala Trp Glu Val Asn Lys Gly 100 105 110 Thr Asn Cys Val Thr Ser Tyr Leu Thr Asp Cys Glu Thr Gln Leu Ser 115 120 125 Gln Ala Pro Arg Gln Gly Leu Leu Tyr Gly Val Pro Val Ser Leu Lys 130 135 140 Glu Cys Phe Ser Tyr Lys Gly His Ala Ser Thr Leu Gly Leu Ser Leu 145 150 155 160 Asn Glu Gly Val Thr Ser Glu Ser Asp Cys Val Val Val Gln Val Leu 165 170 175 Lys Leu Gln Gly Ala Val Pro Phe Val His Thr Asn Val Pro Gln Ser 180 185 190 Met Leu Ser Tyr Asp Cys Ser Asn Pro Leu Phe Gly Gln Thr Met Asn 195 200 205 Pro Trp Lys Pro Ser Lys Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly 210 215 220 Ala Leu Ile Gly Ser Gly Gly Ser Pro Leu Gly Leu Gly Thr Asp Ile 225 230 235 240 Gly Gly Ser Ile Arg Phe Pro Ser Ala Phe Cys Gly Ile Cys Gly Leu 245 250 255 Lys Pro Thr Gly Asn Arg Leu Ser Lys Ser Gly Leu Lys Ser Cys Val 260 265 270 Tyr Gly Gln Thr Ala Val Gln Leu Ser Val Gly Pro Met Ala Arg Asp 275 280 285 Val Asp Ser Leu Ala Leu Cys Met Lys Ala Leu Leu Cys Glu Asp Leu 290 295 300 Phe Arg Leu Asp Ser Thr Ile Pro Pro Leu Pro Phe Arg Glu Glu Ile 305 310 315 320 Tyr Arg Ser Ser Arg Pro Leu Arg Val Gly Tyr Tyr Glu Thr Asp Asn 325 330 335 Tyr Thr Met Pro Thr Pro Ala Met Arg Arg Ala Val Met Glu Thr Lys 340 345 350 Gln Ser Leu Glu Ala Ala Gly His Thr Leu Val Pro Phe Leu Pro Asn 355 360 365 Asn Ile Pro Tyr Ala Leu Glu Val Leu Ser Ala Gly Gly Leu Phe Ser 370 375 380 Asp Gly Gly Cys Ser Phe Leu Gln Asn Phe Lys Gly Asp Phe Val Asp 385 390 395 400 Pro Cys Leu Gly Asp Leu Val Leu Val Leu Lys Leu Pro Arg Trp Phe 405 410 415 Lys Lys Leu Leu Ser Phe Leu Leu Lys Pro Leu Phe Pro Arg Leu Ala 420 425 430 Ala Phe Leu Asn Ser Met Cys Pro Arg Ser Ala Glu Lys Leu Trp Glu 435 440 445 Leu Gln His Glu Ile Glu Met Tyr Arg Gln Ser Val Ile Ala Gln Trp 450 455 460 Lys Ala Met Asn Leu Asp Val Val Leu Thr Pro Met Leu Gly Pro Ala 465 470 475 480 Leu Asp Leu Asn Thr Pro Gly Arg Ala Thr Gly Ala Ile Ser Tyr Thr 485 490 495 Val Leu Tyr Asn Cys Leu Asp Phe Pro Ala Gly Val Val Pro Val Thr 500 505 510 Thr Val Thr Ala Glu Asp Asp Ala Gln Met Glu His Tyr Lys Gly Tyr 515 520 525 Phe Gly Asp Met Trp Asp Asn Ile Leu Lys Lys Gly Met Lys Lys Gly 530 535 540 Ile Gly Leu Pro Val Ala Val Gln Cys Val Ala Leu Pro Trp Gln Glu 545 550 555 560 Glu Leu Cys Leu Arg Phe Met Arg Glu Val Glu Arg Leu Met Thr Pro 565 570 575 Glu Lys Arg Pro Ser 580 1959 base pairs nucleic acid double linear cDNA NO NO not provided 41 GATCCCCCGG GCTGCAGGAA TTCCGGGGGA GCTGGTCCAG ACTTGGTTAT GGAGGGCATG 60 TCCTGGGTTT CTGTAGGAGT CCAGGGGAGG GCAGGTGGTT GCCTCTTCCC GTGCTGGGCA 120 TCTCCTCATG GGTGGCAGGA TCCTGAATAG ATGGGGCTGC AGTGCAGAGC GCATGGGCCT 180 TAGGTCCTCC AGAGCTGGGC AGATGAATGA CCCTCAAGAT GGCCGCTTTT CAGGGGTCAT 240 CAGCCGTTCC ACCTCCCGCA TGAACCGCAG ACACAGCTCT TCCTGCCAGG GCAGAGCCAC 300 GCACTGCACA GCCACAGGCA GGCCTATACC CTTTTTCATG CCCTTCTTCA GAATGTTGTC 360 CCACATATCC CCAAAGTAGC CTTTGTAGTG TTCCATCTGG GCATCGTCCT CAGCGGTCAC 420 AGTGGTGACA GGCACCACCC CCGCAGGGAA GTCCAGGCAG TTATAGAGAA CAGTGTAGCT 480 GATAGCCCCT GTGGCTCTGC CCGGTGTGTT CAAATCCAGA GCAGGACCCA GCATGGGGGT 540 TAGCACCACG TCCAAGTTCA TTGCCTTCCA CTGGGCAATG ACGGACTGGC GATACATCTC 600 AATCTCATGC TGCAGTTCCC ACAGCTTTTC GGCTGACCGA GGACACATAC TGTTGAGAAA 660 GGCTGCCAGC CGAGGAAACA GAGGCTTCAG CAGGAAGCTC AGCAGTTTTT TAAACCACCT 720 GGGCAGCTTC AGCACTAAGA CCAGGTCCCC CAAGCAGGGA TCCACAAAGT CGCCTTTGAA 780 GTTTTGGAGA AAAGAGCAGC CACCATCACT GAACAGCCCA CCTGCCGACA GGACCTCCAG 840 GGCATAAGGT ATGTTGTTTG GTAAGAAGGG GACCAGCGTG TGGCCAGCAG CCTCGAGACT 900 CTGCTTGGTC TCCATCACAG CCCTCCTCAT GGCTGGAGTG GGCATGGTGT AGTTGTCAGT 960 TTCATAGTAT CCCACACGAA GGGGTCGAGA ACTTCTGTAG ATCTCCTCCC TGAAGGGCAA 1020 GGGGGGGATG GTGGAGTCCA AGCGGAACAA ATCCTCACAA AGTAGGGCTT TCATGCACAA 1080 TGCCAGGCTA TCCACATCCC GTGCCATGGG GCCAACAGAA AGCTGCACTG CTGTCTGTCC 1140 ATAAACACAG CTCTTCAGGC CACTCTTGCT GAGGCGGTTC CCAGTAGGCT TGAGGCCACA 1200 GATGCCACAG AAGGCAGAAG GGAACCGGAT GCTGCCGCCG ATGTCAGTGC CTAAACCCAG 1260 AGGGGAGCCT CCAGATCCAA TGAGAGCCCC CTCACCCCCT GAGGAACCTC CTGGACTCTT 1320 GGAGGGCTTC CACGGGTTCA TGGTCTGGCC AAAGAGGGGG TTACTGCAGT CATAGCTTAG 1380 CATGGACTGG GGGACGTTGG TGTGCACAAA GGGCACAGCT CCCTGCAGCT TCAGTACCTG 1440 CACCACCACA CAGTCACTCT CCGATGTCAC ACCCTCGTTC AAACTTAAGC CCAGTGTGGA 1500 AGCATGGCCC TTGTAGCTGA AGCATTCCTT GAGGCTCACG GGGACGCCAT AGAGCAGGCC 1560 CTGCCGTGGG GCCTGGGACA GCTGAGTCTC ACAGTCAGTC AGATAGGAGG TCACACAGTT 1620 GGTCCCTTTG TTCACTTCCC AGGCCTTTCC CAGGTAGGTA AAGAGCACAG CTTCTGGGGA 1680 CAGTTCCCCA CTCTGTAACT TCTGTACCAG TTGGAGCAGG GGCAGAGCCA GCAAGGCCTC 1740 TGAATCCAGG TCAGGATTCT GCAGCCGGAA GCGCTGCACC GCCTTGTCCA TGGTCTCCAG 1800 GCCGGCTCGC TGCTTCTGCC GCGCCCTGGT CACCGCGCCC CGGGCGGTCT GGCTCCTGGT 1860 CCATCGCAGG ACCACCGCCG CCGACAGCAA GCTGCAGGCT AGGCAAACCC CGGAGAGTCC 1920 AGACAGCGCG GTCCACACTT CGCTCAGCAC CATGACCCA 1959 2045 base pairs nucleic acid double linear cDNA NO NO not provided CDS 3..1775 42 TG CCG GGC GGT AGG CAG CAG CAG GCT GAA GGG ATC ATG GTG CAG TAC 47 Pro Gly Gly Arg Gln Gln Gln Ala Glu Gly Ile Met Val Gln Tyr 1 5 10 15 GAG CTG TGG GCC GCG CTG CCT GGC GCC TCC GGG GTC GCC CTG GCC TGC 95 Glu Leu Trp Ala Ala Leu Pro Gly Ala Ser Gly Val Ala Leu Ala Cys 20 25 30 TGC TTC GTG GCG GCG GCC GTG GCC CTG CGC TGG TCC GGG CGC CGG ACG 143 Cys Phe Val Ala Ala Ala Val Ala Leu Arg Trp Ser Gly Arg Arg Thr 35 40 45 GCG CGG GGC GCG GTG GTC CGG GCG CGA CAG AAG CAG CGA GCG GGC CTG 191 Ala Arg Gly Ala Val Val Arg Ala Arg Gln Lys Gln Arg Ala Gly Leu 50 55 60 GAG AAC ATG GAC AGG GCG GCG CAG CGC TTC CGG CTC CAG AAC CCA GAC 239 Glu Asn Met Asp Arg Ala Ala Gln Arg Phe Arg Leu Gln Asn Pro Asp 65 70 75 CTG GAC TCA GAG GCG CTG CTA GCC CTG CCC CTG CCT CAG CTG GTG CAG 287 Leu Asp Ser Glu Ala Leu Leu Ala Leu Pro Leu Pro Gln Leu Val Gln 80 85 90 95 AAG TTA CAC AGT AGA GAG CTG GCC CCT GAG GCC GTG CTC TTC ACC TAT 335 Lys Leu His Ser Arg Glu Leu Ala Pro Glu Ala Val Leu Phe Thr Tyr 100 105 110 GTG GGA AAG GCC TGG GAA GTG AAC AAA GGG ACC AAC TGT GTG ACC TCC 383 Val Gly Lys Ala Trp Glu Val Asn Lys Gly Thr Asn Cys Val Thr Ser 115 120 125 TAT CTG GCT GAC TGT GAG ACT CAG CTG TCT CAG GCC CCA AGG CAG GGC 431 Tyr Leu Ala Asp Cys Glu Thr Gln Leu Ser Gln Ala Pro Arg Gln Gly 130 135 140 CTG CTC TAT GGC GTC CCT GTG AGC CTC AAG GAG TGC TTC ACC TAC AAG 479 Leu Leu Tyr Gly Val Pro Val Ser Leu Lys Glu Cys Phe Thr Tyr Lys 145 150 155 GGC CAG GAC TCC ACG CTG GGC TTG AGC CTG AAT GAA GGG GTG CCG GCG 527 Gly Gln Asp Ser Thr Leu Gly Leu Ser Leu Asn Glu Gly Val Pro Ala 160 165 170 175 GAG TGC GAC AGC GTA GTG GTG CAT GTG CTG AAG CTG CAG GGT GCC GTG 575 Glu Cys Asp Ser Val Val Val His Val Leu Lys Leu Gln Gly Ala Val 180 185 190 CCC TTC GTG CAC ACC AAT GTT CCA CAG TCC ATG TTC AGC TAT GAC TGC 623 Pro Phe Val His Thr Asn Val Pro Gln Ser Met Phe Ser Tyr Asp Cys 195 200 205 AGT AAC CCC CTC TTT GGC CAG ACC GTG AAC CCA TGG AAG TCC TCC AAA 671 Ser Asn Pro Leu Phe Gly Gln Thr Val Asn Pro Trp Lys Ser Ser Lys 210 215 220 AGC CCA GGG GGC TCC TCA GGG GGT GAA GGG GCC CTC ATC GGG TCT GGA 719 Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly Ser Gly 225 230 235 GGC TCC CCC CTG GGC TTA GGC ACT GAT ATC GGA GGC AGC ATC CGC TTC 767 Gly Ser Pro Leu Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Phe 240 245 250 255 CCC TCC TCC TTC TGC GGC ATC TGC GGC CTC AAG CCC ACA GGG AAC CGC 815 Pro Ser Ser Phe Cys Gly Ile Cys Gly Leu Lys Pro Thr Gly Asn Arg 260 265 270 CTC AGC AAG AGT GGC CTG AAG GGC TGT GTC TAT GGA CAG GAG GCA GTG 863 Leu Ser Lys Ser Gly Leu Lys Gly Cys Val Tyr Gly Gln Glu Ala Val 275 280 285 CGT CTC TCC GTG GGC CCC ATG GCC CGG GAC GTG GAG AGC CTG GCA CTG 911 Arg Leu Ser Val Gly Pro Met Ala Arg Asp Val Glu Ser Leu Ala Leu 290 295 300 TGC CTG CGA GCC CTG CTG TGC GAG GAC ATG TTC CGC TTG GAC CCC ACT 959 Cys Leu Arg Ala Leu Leu Cys Glu Asp Met Phe Arg Leu Asp Pro Thr 305 310 315 GTG CCT CCC TTG CCC TTC AGA GAA GAG GTC TAC ACC AGC TCT CAG CCC 1007 Val Pro Pro Leu Pro Phe Arg Glu Glu Val Tyr Thr Ser Ser Gln Pro 320 325 330 335 CTG CGT GTG GGG TAC TAT GAG ACT GAC AAC TAT ACC ATG CCC TCC CCG 1055 Leu Arg Val Gly Tyr Tyr Glu Thr Asp Asn Tyr Thr Met Pro Ser Pro 340 345 350 GCC ATG AGG CGG GCC GTG CTG GAG ACC AAA CAG AGC CTT GAG GCT GCG 1103 Ala Met Arg Arg Ala Val Leu Glu Thr Lys Gln Ser Leu Glu Ala Ala 355 360 365 GGG CAC ACG CTG GTT CCC TTC TTG CCA AGC AAC ATA CCC CAT GCT CTG 1151 Gly His Thr Leu Val Pro Phe Leu Pro Ser Asn Ile Pro His Ala Leu 370 375 380 GAG ACC CTG TCA ACA GGT GGG CTC TTC AGT GAT GGT GGC CAC ACC TTC 1199 Glu Thr Leu Ser Thr Gly Gly Leu Phe Ser Asp Gly Gly His Thr Phe 385 390 395 CTA CAG AAC TTC AAA GGT GAT TTC GTG GAC CCC TGC CTG GGG GAC CTG 1247 Leu Gln Asn Phe Lys Gly Asp Phe Val Asp Pro Cys Leu Gly Asp Leu 400 405 410 415 GTC TCA ATT CTG AAG CTT CCC CAA TGG CTT AAA GGA CTG CTG GCC TTC 1295 Val Ser Ile Leu Lys Leu Pro Gln Trp Leu Lys Gly Leu Leu Ala Phe 420 425 430 CTG GTG AAG CCT CTG CTG CCA AGG CTG TCA GCT TTC CTC AGC AAC ATG 1343 Leu Val Lys Pro Leu Leu Pro Arg Leu Ser Ala Phe Leu Ser Asn Met 435 440 445 AAG TCT CGT TCG GCT GGA AAA CTC TGG GAA CTG CAG CAC GAG ATC GAG 1391 Lys Ser Arg Ser Ala Gly Lys Leu Trp Glu Leu Gln His Glu Ile Glu 450 455 460 GTG TAC CGC AAA ACC GTG ATT GCC CAG TGG AGG GCG CTG GAC CTG GAT 1439 Val Tyr Arg Lys Thr Val Ile Ala Gln Trp Arg Ala Leu Asp Leu Asp 465 470 475 GTG GTG CTG ACC CCC ATG CTG GCC CCT GCT CTG GAC TTG AAT GCC CCA 1487 Val Val Leu Thr Pro Met Leu Ala Pro Ala Leu Asp Leu Asn Ala Pro 480 485 490 495 GGC AGG GCC ACA GGG GCC GTC AGC TAC ACT ATG CTG TAC AAC TGC CTG 1535 Gly Arg Ala Thr Gly Ala Val Ser Tyr Thr Met Leu Tyr Asn Cys Leu 500 505 510 GAC TTC CCT GCA GGG GTG GTG CCT GTC ACC ACG GTG ACT GCT GAG GAC 1583 Asp Phe Pro Ala Gly Val Val Pro Val Thr Thr Val Thr Ala Glu Asp 515 520 525 GAG GCC CAG ATG GAA CAT TAC AGG GGC TAC TTT GGG GAT ATC TGG GAC 1631 Glu Ala Gln Met Glu His Tyr Arg Gly Tyr Phe Gly Asp Ile Trp Asp 530 535 540 AAG ATG CTG CAG AAG GGC ATG AAG AAG AGT GTG GGG CTG CCG GTG GCC 1679 Lys Met Leu Gln Lys Gly Met Lys Lys Ser Val Gly Leu Pro Val Ala 545 550 555 GTG CAG TGT GTG GCT CTG CCC TGG CAA GAA GAG TTG TGT CTG CGG TTC 1727 Val Gln Cys Val Ala Leu Pro Trp Gln Glu Glu Leu Cys Leu Arg Phe 560 565 570 575 ATG CGG GAG GTG GAG CGA CTG ATG ACC CCT GAA AAG CAG TCA TCC TGATGGCT1782 Met Arg Glu Val Glu Arg Leu Met Thr Pro Glu Lys Gln Ser Ser 580 585 590 GGCTCCAGAG GACCTGAGAC TCACACTCTC TGCAGCCCAG CCTAGTCAGG GCACAGCTGC 1842 CCTGCTGCCA CAGCAAGGAA ATGTCCTGCA TGGGGCAGAG GCTTCCGTGT CCTCTCCCCC 1902 AACCCCCTGC AAGAAGCGCC GACTCCCTGA GTCTGGACCT CCATCCCTGC TCTGGTCCCC 1962 TCTCTTCGTC CTGATCCCTC CACCCCCATG TGGCAGCCCA TGGGTATGAC ATAGGCCAAG 2022 GCCCAACTAA CAGCCCCGGA ATT 2045 590 amino acids amino acid linear protein not provided 43 Pro Gly Gly Arg Gln Gln Gln Ala Glu Gly Ile Met Val Gln Tyr Glu 1 5 10 15 Leu Trp Ala Ala Leu Pro Gly Ala Ser Gly Val Ala Leu Ala Cys Cys 20 25 30 Phe Val Ala Ala Ala Val Ala Leu Arg Trp Ser Gly Arg Arg Thr Ala 35 40 45 Arg Gly Ala Val Val Arg Ala Arg Gln Lys Gln Arg Ala Gly Leu Glu 50 55 60 Asn Met Asp Arg Ala Ala Gln Arg Phe Arg Leu Gln Asn Pro Asp Leu 65 70 75 80 Asp Ser Glu Ala Leu Leu Ala Leu Pro Leu Pro Gln Leu Val Gln Lys 85 90 95 Leu His Ser Arg Glu Leu Ala Pro Glu Ala Val Leu Phe Thr Tyr Val 100 105 110 Gly Lys Ala Trp Glu Val Asn Lys Gly Thr Asn Cys Val Thr Ser Tyr 115 120 125 Leu Ala Asp Cys Glu Thr Gln Leu Ser Gln Ala Pro Arg Gln Gly Leu 130 135 140 Leu Tyr Gly Val Pro Val Ser Leu Lys Glu Cys Phe Thr Tyr Lys Gly 145 150 155 160 Gln Asp Ser Thr Leu Gly Leu Ser Leu Asn Glu Gly Val Pro Ala Glu 165 170 175 Cys Asp Ser Val Val Val His Val Leu Lys Leu Gln Gly Ala Val Pro 180 185 190 Phe Val His Thr Asn Val Pro Gln Ser Met Phe Ser Tyr Asp Cys Ser 195 200 205 Asn Pro Leu Phe Gly Gln Thr Val Asn Pro Trp Lys Ser Ser Lys Ser 210 215 220 Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly Ser Gly Gly 225 230 235 240 Ser Pro Leu Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Phe Pro 245 250 255 Ser Ser Phe Cys Gly Ile Cys Gly Leu Lys Pro Thr Gly Asn Arg Leu 260 265 270 Ser Lys Ser Gly Leu Lys Gly Cys Val Tyr Gly Gln Glu Ala Val Arg 275 280 285 Leu Ser Val Gly Pro Met Ala Arg Asp Val Glu Ser Leu Ala Leu Cys 290 295 300 Leu Arg Ala Leu Leu Cys Glu Asp Met Phe Arg Leu Asp Pro Thr Val 305 310 315 320 Pro Pro Leu Pro Phe Arg Glu Glu Val Tyr Thr Ser Ser Gln Pro Leu 325 330 335 Arg Val Gly Tyr Tyr Glu Thr Asp Asn Tyr Thr Met Pro Ser Pro Ala 340 345 350 Met Arg Arg Ala Val Leu Glu Thr Lys Gln Ser Leu Glu Ala Ala Gly 355 360 365 His Thr Leu Val Pro Phe Leu Pro Ser Asn Ile Pro His Ala Leu Glu 370 375 380 Thr Leu Ser Thr Gly Gly Leu Phe Ser Asp Gly Gly His Thr Phe Leu 385 390 395 400 Gln Asn Phe Lys Gly Asp Phe Val Asp Pro Cys Leu Gly Asp Leu Val 405 410 415 Ser Ile Leu Lys Leu Pro Gln Trp Leu Lys Gly Leu Leu Ala Phe Leu 420 425 430 Val Lys Pro Leu Leu Pro Arg Leu Ser Ala Phe Leu Ser Asn Met Lys 435 440 445 Ser Arg Ser Ala Gly Lys Leu Trp Glu Leu Gln His Glu Ile Glu Val 450 455 460 Tyr Arg Lys Thr Val Ile Ala Gln Trp Arg Ala Leu Asp Leu Asp Val 465 470 475 480 Val Leu Thr Pro Met Leu Ala Pro Ala Leu Asp Leu Asn Ala Pro Gly 485 490 495 Arg Ala Thr Gly Ala Val Ser Tyr Thr Met Leu Tyr Asn Cys Leu Asp 500 505 510 Phe Pro Ala Gly Val Val Pro Val Thr Thr Val Thr Ala Glu Asp Glu 515 520 525 Ala Gln Met Glu His Tyr Arg Gly Tyr Phe Gly Asp Ile Trp Asp Lys 530 535 540 Met Leu Gln Lys Gly Met Lys Lys Ser Val Gly Leu Pro Val Ala Val 545 550 555 560 Gln Cys Val Ala Leu Pro Trp Gln Glu Glu Leu Cys Leu Arg Phe Met 565 570 575 Arg Glu Val Glu Arg Leu Met Thr Pro Glu Lys Gln Ser Ser 580 585 590 2045 base pairs nucleic acid double linear cDNA NO NO not provided 44 AATTCCGGGG CTGTTAGTTG GGCCTTGGCC TATGTCATAC CCATGGGCTG CCACATGGGG 60 GTGGAGGGAT CAGGACGAAG AGAGGGGACC AGAGCAGGGA TGGAGGTCCA GACTCAGGGA 120 GTCGGCGCTT CTTGCAGGGG GTTGGGGGAG AGGACACGGA AGCCTCTGCC CCATGCAGGA 180 CATTTCCTTG CTGTGGCAGC AGGGCAGCTG TGCCCTGACT AGGCTGGGCT GCAGAGAGTG 240 TGAGTCTCAG GTCCTCTGGA GCCAGAGCCA TCAGGATGAC TGCTTTTCAG GGGTCATCAG 300 TCGCTCCACC TCCCGCATGA ACCGCAGACA CAACTCTTCT TGCCAGGGCA GAGCCACACA 360 CTGCACGGCC ACCGGCAGCC CCACACTCTT CTTCATGCCC TTCTGCAGCA TCTTGTCCCA 420 GATATCCCCA AAGTAGCCCC TGTAATGTTC CATCTGGGCC TCGTCCTCAG CAGTCACCGT 480 GGTGACAGGC ACCACCCCTG CAGGGAAGTC CAGGCAGTTG TACAGCATAG TGTAGCTGAC 540 GGCCCCTGTG GCCCTGCCTG GGGCATTCAA GTCCAGAGCA GGGGCCAGCA TGGGGGTCAG 600 CACCACATCC AGGTCCAGCG CCCTCCACTG GGCAATCACG GTTTTGCGGT ACACCTCGAT 660 CTCGTGCTGC AGTTCCCAGA GTTTTCCAGC CGAACGAGAC TTCATGTTGC TGAGGAAAGC 720 TGACAGCCTT GGCAGCAGAG GCTTCACCAG GAAGGCCAGC AGTCCTTTAA GCCATTGGGG 780 AAGCTTCAGA ATTGAGACCA GGTCCCCCAG GCAGGGGTCC ACGAAATCAC CTTTGAAGTT 840 CTGTAGGAAG GTGTGGCCAC CATCACTGAA GAGCCCACCT GTTGACAGGG TCTCCAGAGC 900 ATGGGGTATG TTGCTTGGCA AGAAGGGAAC CAGCGTGTGC CCCGCAGCCT CAAGGCTCTG 960 TTTGGTCTCC AGCACGGCCC GCCTCATGGC CGGGGAGGGC ATGGTATAGT TGTCAGTCTC 1020 ATAGTACCCC ACACGCAGGG GCTGAGAGCT GGTGTAGACC TCTTCTCTGA AGGGCAAGGG 1080 AGGCACAGTG GGGTCCAAGC GGAACATGTC CTCGCACAGC AGGGCTCGCA GGCACAGTGC 1140 CAGGCTCTCC ACGTCCCGGG CCATGGGGCC CACGGAGAGA CGCACTGCCT CCTGTCCATA 1200 GACACAGCCC TTCAGGCCAC TCTTGCTGAG GCGGTTCCCT GTGGGCTTGA GGCCGCAGAT 1260 GCCGCAGAAG GAGGAGGGGA AGCGGATGCT GCCTCCGATA TCAGTGCCTA AGCCCAGGGG 1320 GGAGCCTCCA GACCCGATGA GGGCCCCTTC ACCCCCTGAG GAGCCCCCTG GGCTTTTGGA 1380 GGACTTCCAT GGGTTCACGG TCTGGCCAAA GAGGGGGTTA CTGCAGTCAT AGCTGAACAT 1440 GGACTGTGGA ACATTGGTGT GCACGAAGGG CACGGCACCC TGCAGCTTCA GCACATGCAC 1500 CACTACGCTG TCGCACTCCG CCGGCACCCC TTCATTCAGG CTCAAGCCCA GCGTGGAGTC 1560 CTGGCCCTTG TAGGTGAAGC ACTCCTTGAG GCTCACAGGG ACGCCATAGA GCAGGCCCTG 1620 CCTTGGGGCC TGAGACAGCT GAGTCTCACA GTCAGCCAGA TAGGAGGTCA CACAGTTGGT 1680 CCCTTTGTTC ACTTCCCAGG CCTTTCCCAC ATAGGTGAAG AGCACGGCCT CAGGGGCCAG 1740 CTCTCTACTG TGTAACTTCT GCACCAGCTG AGGCAGGGGC AGGGCTAGCA GCGCCTCTGA 1800 GTCCAGGTCT GGGTTCTGGA GCCGGAAGCG CTGCGCCGCC CTGTCCATGT TCTCCAGGCC 1860 CGCTCGCTGC TTCTGTCGCG CCCGGACCAC CGCGCCCCGC GCCGTCCGGC GCCCGGACCA 1920 GCGCAGGGCC ACGGCCGCCG CCACGAAGCA GCAGGCCAGG GCGACCCCGG AGGCGCCAGG 1980 CAGCGCGGCC CACAGCTCGT ACTGCACCAT GATCCCTTCA GCCTGCTGCT GCCTACCGCC 2040 CGGCA 2045 26 base pairs nucleic acid single linear cDNA NO NO not provided 45 GCGGTACCAT GCGATGGACC GGGCGC 26 18 base pairs nucleic acid single linear cDNA NO NO not provided 46 GGTCTGGCCA AAGAGAGG 18 32 amino acids amino acid linear protein internal not provided 47 Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Ala Gly Gly Gly Ser 1 5 10 15 Leu Leu Gly Ile Gly Ser Asp Val Ala Gly Ser Ile Arg Leu Pro Ser 20 25 30 32 amino acids amino acid linear protein internal not provided 48 Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly Ala Gly Gly Ser 1 5 10 15 Leu Ile Gly Ile Gly Thr Asp Val Gly Gly Ser Val Arg Ile Pro Cys 20 25 30 32 amino acids amino acid linear protein internal not provided 49 Gly Gly Ser Ser Gly Gly Glu Ser Ala Leu Ile Ser Ala Asp Gly Ser 1 5 10 15 Leu Leu Gly Ile Gly Gly Asp Val Gly Gly Ser Ile Arg Ile Pro Cys 20 25 30 32 amino acids amino acid linear protein internal not provided 50 Gly Gly Ser Ser Gly Gly Glu Gly Ser Leu Ile Gly Ala His Gly Ser 1 5 10 15 Leu Leu Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Ile Pro Ser 20 25 30 32 amino acids amino acid linear protein internal not provided 51 Gly Gly Ser Ser Gly Gly Glu Gly Ala Ile Val Gly Ile Arg Gly Gly 1 5 10 15 Val Ile Gly Val Gly Thr Asp Ile Gly Gly Ser Ile Asp Val Pro Ala 20 25 30 32 amino acids amino acid linear protein internal not provided 52 Gly Gly Ser Ser Gly Gly Val Ala Ala Ala Val Ala Ser Arg Leu Met 1 5 10 15 Leu Gly Gly Ile Gly Thr Asp Thr Gly Ala Ser Val Arg Leu Pro Ala 20 25 30 32 amino acids amino acid linear protein internal not provided 53 Gly Gly Ser Ser Gly Gly Val Ala Ala Ala Val Ala Ser Gly Ile Val 1 5 10 15 Pro Leu Ser Val Gly Thr Asp Thr Gly Gly Ser Ile Arg Ile Pro Ala 20 25 30 819 base pairs nucleic acid double linear cDNA NO NO not provided 54 CCAGGAGGTT CCTCAGGGGG TGAGGGGGCT CTCATTGGAT CTGGAGGTTC CCCTCTGGGT 60 TTAGGCACTG ACATTGGCGG CAGCATCCGG TTCCCTTCTG CCTTCTGCGG CATCTGTGGC 120 CTCAAGCCTA CTGGCAACCG CCTCAGCAAG AGTGGCCTGA AGGGCTGTGT CTATGGACAG 180 ACGGCAGTGC AGCTTTCTCT TGGCCCCATG GCCCGGGATG TGGAGAGCCT GGCGCTATGC 240 CTGAAAGCTC TACTGTGTGA GCACTTGTTC ACCTTGGACC CTACCGTGCC TCCCTTTCCC 300 TTCAGAGAGG AGGTCTATAG AAGTTCTAGA CCCCTGCGTG TGGGGTACTA TGAGACTGAC 360 AACTATACCA TGCCCAGCCC AGCTATGAGG AGGGCTCTGA TAGAGACCAA GCAGAGACTT 420 GAGGCTGCTG GCCACACGCT GATTCCCTTC TTACCCAACA ACATACCCTA CGCCCTGGAG 480 GTCCTGTCTG CGGGCGGCCT GTTCAGTGAC GGTGGCCGCA GTTTTCTCCA AAACTTCAAA 540 GGTGACTTTG TGGATCCCTG CTTGGGAGAC CTGATCTTAA TTCTGAGGCT GCCCAGCTGG 600 TTTAAAAGAC TGCTGAGCCT CCTGCTGAAG CCTCTGTTTC CTCGGCTGGC AGCCTTTCTC 660 AACAGTATGC GTCCTCGGTC AGCTGAAAAG CTGTGGAAAC TGCAGCATGA GATTGAGATG 720 TATCGCCAGT CTGTGATTGC CCAGTGGAAA GCGATGAACT TGGATGTGCT GCTGACCCCN 780 ATGYTNGGNC CNGCNYTNGA YYTNAAYACN CCNGGNMGN 819 

What is claimed is:
 1. An isolated and purified polypeptide that has the amino acid residue sequence shown in SEQ ID NO
 36. 2. The polypeptide of claim 1 that hydrolyzes myristic amide, palmitic amide and stearic amide.
 3. An isolated and purified polypeptide that has the amino acid residue sequence shown in SEQ ID NO
 40. 4. The polypeptide of claim 3 that hydrolyzes myristic amide, palmitic amide and stearic amide.
 5. An isolated and purified polypeptide that has the amino acid residue sequence shown in SEQ ID NO
 43. 6. The polypeptide of claim 5 that hydrolyzes myristic amide, palmitic amide and stearic amide. 